Fast and exhaustive method for selecting a prey polypeptide interacting with a bait polypeptide of interest: application to the construction of maps of interactors polypeptides

ABSTRACT

The present invention is directed to a method for selecting a prey polypeptide that is able to interact with a bait polypeptide of interest, to a prey polynucleotide encoding the prey polypeptide as well as to the prey polypeptide itself. The invention also concerns plasmids used for performing the method of the invention as well as prokaryotic or eukaryotic recombinant host organisms containing such plasmids and also a collection of said recombinant host organisms consisting in a DNA library, such as a collection of recombinant haploid  Saccharomyces cerevisiae . Finally, the invention is also directed to a technical medium containing the whole information concerning the interactions between metabolically related bait and prey polypeptides and/or polynucleotides coding for bait and prey polypeptides.

[0001] The present invention is directed to a method for selecting aprey polypeptide that is able to interact with a bait polypeptide ofinterest, to a prey polynucleotide encoding the prey polypeptide as wellas to the prey polypeptide itself. The invention also concerns plasmidsused for performing the method of the invention as well as prokaryoticor eukaryotic recombinant host organisms containing such plasmids andalso a collection of said recombinant host organisms consisting in a DNAlibrary, such as a collection of recombinant haploid Saccharomycescerevisiae. Finally, the invention is also directed to a technicalmedium containing the whole information concerning the interactionsbetween metabolically related bait and prey polypeptides and/orpolynucleotides coding for bait and prey polypeptides.

BACKGROUND OF THE INVENTION

[0002] Most biological processes involve specific protein-proteininteractions. General methodologies to identify interacting proteins orto study these interactions have been extensively developed. Among them,the yeast two-hybrid system currently represents the most powerful invivo approach to screen for polypeptides that could bind to a giventarget protein. Originally developed by Fields and coworkers [(Fields etal., 1989; Chien et al., 1991). Two U.S. Pat. No. 5,283,173 granted onFeb. 1, 1994 (Fields, S. & Song, O.) and U.S. Pat. No. 5,468,614 grantedon Nov. 21, 1995 (Fields, S. & Song, O.) herein incorporated byreference], the two-hybrid system utilizes hybrid genes to detectprotein-protein interactions by means of direct activation of areporter-gene expression (Allen et al., 1995; Transy et al., 1995). Inessence, the two putative protein partners are genetically fused to theDNA-binding domain of a transcription factor and to a transcriptionalactivation domain, respectively. A productive interaction between thetwo proteins of interest will bring the transcriptional activationdomain in the proximity of the DNA-binding domain and will triggerdirectly the transcription of an adjacent reporter gene (usually lacZ ora nutritional marker) giving a screenable phenotype. The transcriptioncan be activated through the use of two functional domains of atranscription factor: a domain that recognizes and binds to a specificsite on the DNA and a domain that is necessary for activation, asreported by Keegan et al. (1986) and Ma et al. (1987).

[0003] Recently, Rossi et al. (1997) described a different approach, amammalian “two-hybrid” system, which uses β-galactosidasecomplementation (Ullmann et al., 1968) to monitor protein-proteininteractions in intact eukaryotic cells.

[0004] The number of genome sequences of prokaryotic as well aseukaryotic host organisms available is increasing exponentially andthere is a great need for new tools directed to the functional andglobal study of these newly characterized complete or partial genomes.As an illustrative example, the genome of the yeast Saccharomycescerevisiae is now completely sequenced (Goffeau et al., 1996). Despitethe tremendous and successful genetic work in past years, 60% of yeastgenes have no assigned function and half of those encode putativeproteins without any homology with known proteins (Dujon, 1996). Inyeast, genetic analyses, such as suppressor or synthetic lethal screens,have suggested many functional links between gene products, some ofwhich have later been confirmed by biochemical means. All together,these approaches have led to a rather extensive knowledge of definedbiochemical pathways. However, the integration of these pathways in thecomplexity of a living cell remains to be accomplished. To explore theintegrative functions and find the molecular factors sustaining them,some authors have attempted to design new screens. However, thesescreens are usually very specific and cannot apply directly to manydifferent cellular functions. In addition, few yeast genes areessential, leading to an additional difficulty for genetic screens.Other approaches developed by cellular biologists seek to preciselylocalize proteins within the cell. The assumption is that colocalizationof factors is indicative of functional interactions. This approach hasbeen very successful, despite the fact that it is usually very elaborateand is rarely considered practicable for a systematic approach (Burns etal., 1994).

[0005] Bartel et al. (1996) extended the approach of the typicaltwo-hybrid system consisting in a known protein that forms a part of aDNA-binding domain hybrid, assayed against a library of all possibleproteins present as transcriptional activation domain hybrids, using thegenome of bacteriophage T7, such that a second library of all possibleproteins fused to the DNA-binding domain to be analyzed. Thisgenome-wide approach to the two-hybrid searches has identified 25interactions among the proteins of T7.

[0006] However, the currently available two-hybrid methodology is notsuitable for a large scale project without specific methodologicalimprovements. Although the two-hybrid strategy has been a major tool inproving protein:protein interactions between factors known to befunctionally related (Fields and Song, 1989), its use for exhaustive andreliable search for unknown partners of a given protein is moreproblematic. Thus, in most cases, the two-hybrid screen constitutes aninitial screen in which many different interactions are found. Among theidentified candidates, only some of them are favored due to theirappealing sequence. Subsequent functional assays are required forestablishing their possible biological significance. For these reasons,the two-hybrid methodology has been considered a difficult, if notmisleading experimental approach for screening.

[0007] Finley et al. (1994) or Bendixen et al. (1994) have describedtwo-hybrid systems including a step of mating yeast cell colonies byreplica-plating diploids, that is to say by mating colonies of yeastcells. Finley et al. (1994) have, in a first step, selected specificinserts of a DNA library using, as selection criteria, the probabilityfor a specific insert contained in the library to comprise a largecoding region of an ORF or to contain a coding region associated with aspecific biological function. For example, Finley et al. (1994) havemade a collection of strains, each of which expressed a different bait(in fact two cyclin dependent kinases [Cdks], namely DmCdc2 andDmCdc2c), and mated them, by replica-plating, with test strains thatcontained different activation-tagged Cdis (Cyclin-dependent kinaseinteractors). Then, each of the selected baits was used as a bait inorder to screen the prey Cdis of the DNA library. Examination of theresulting interaction matrices showed that each Cdi (preys) associatesspecifically with a distinct spectrum of Cdks (baits).

[0008] Despite the fact that these authors state that their resultssuggest a number of applications of their method to geneticcharacterization of larger sets of proteins, it must be pointed out thatthese screening experiments of prior art lead to the constitution ofinteractor polypeptide matrices restricted, as the numerous two-hybridsystems of prior art, by the initial choice of the potentiallyinteresting polynucleotide inserts initially identified and/or initiallyselected in the DNA library.

[0009] Moreover, the replica-plating step that makes use of yeast cellcolonies does not allow the mating of numerous different recombinantyeast cell colonies in a single culture dish, thus rendering veryfastidious, or even materially impossible the study of potentialinteractions between a given bait polypeptide and a wide collection ofprey polypeptides, such as a collection of prey polypeptides encoded bypolynucleotides originating from the whole genome of an organism such asa bacterial, viral or yeast organism.

SUMMARY OF THE INVENTION

[0010] The aim of the present invention is to provide a new method forselecting a polynucleotide encoding a prey polypeptide in a two-hybridscreening system, said method making use of mating recombinant haploidyeast cells instead of recombinant yeast cell colonies, said methodproviding significant advantages over prior art.

[0011] Thus, the present invention provides a method for selecting apolynucleotide encoding a prey polypeptide, said prey polypeptide beingable to interact with a bait polypeptide, comprising the steps of:

[0012] a) subjecting a bait polynucleotide encoding the baitpolypeptide, to a two-hybrid screening method, wherein said two-hybridscreening method comprises a step of mating at least one first haploidrecombinant yeast cell containing the prey polynucleotide to be assayedwith a second haploid recombinant yeast cell containing the baitpolynucleotide, provided that one haploid yeast cell among the firstrecombinant yeast cell or the second recombinant yeast cell alsocontains at least one detectable gene that is activated by a polypeptideincluding a transcriptional activation domain;

[0013] b) selecting the recombinant diploid yeast cell obtained at stepa) for which the detectable gene has been expressed to a degree greaterthan expression in the absence of interaction between the baitpolypeptide and the prey polypeptide;

[0014] c) optionally characterizing the prey polynucleotide contained ineach diploid yeast cell selected at step b).

[0015] By a bait polynucleotide according to the present invention, itis intended a chimeric polynucleotide encoding a chimeric polypeptidecomprising i) a DNA-binding domain that recognizes a binding site on adetectable gene that is contained in a host organism and ii) apolypeptide that is to be tested for interaction with at least one preypolypeptide.

[0016] By a prey polynucleotide according to the present invention, itis intended a chimeric polynucleotide encoding a chimeric polypeptidecomprising i) a transcriptional activation domain and ii) a polypeptidethat is to be tested for interaction with a bait polypeptide.

[0017] Among the numerous improvements brought by the method of theinvention over the existing screening systems, said method:

[0018] i) allows, in a single step, the screening of far more preypolynucleotides with a given bait polynucleotide than the prior artsystems because the mating is performed between haploid yeast cells andnot between yeast cells and a yeast cell colony (or between yeastcolonies);

[0019] ii) As a consequence of i), the method allows the whole screeningof a DNA library without the need of a first step consisting ofselecting the potentially interesting polynucleotide inserts containedtherein and allows an objective analysis of the potential interactorpolypeptides;

[0020] iii) because the mating step is performed between haploidrecombinant yeast cells and not between recombinant yeast cells and ayeast cell colony, and thus because the mating step does not take intoaccount the former differential growth properties of differentrecombinant yeast cell colonies, the method of the invention is far moreexhaustive as well as reproducible than the conventional two-hybridscreening systems. Moreover, an efficient mating step of a shortduration that is performed between individual recombinant haploid yeastcells have two other advantages, namely a) there is a high percentage ofrecombinant diploid yeast cells and not only several diploid recombinantyeast cells dispersed in a single colony and b) the short period of time(less than 5 hours) that is necessary for mating the two populations ofhaploid yeast cells is not sufficient for a significant growth of thehaploid colonies which have not successfully undergone the mating step,nor the doubling of the recombinant diploid yeast cells before plating.

[0021] As another advantageous characteristic of the matin, stepaccording to the two-hybrid screening method of the invention, theinventors have adjusted the experimental protocol in order to obtain upto a 50% increase in the efficiency of the mating procedure, thispercentage being expressed as a ratio of diploid cells generated, andnot as a ratio of recombinant colonies, such as expressed in prior artmating experiments.

[0022] The above-described characteristics of the two-hybrid screeningmethod of the invention leads to a nearly perfect standardization of thediploid yeast cell population under testing. In other words, the wholecharacteristics of the DNA library used as starting material areperfectly reflected in the resulting recombinant diploid yeast cellpopulation after mating. This is why the present two-hybrid screeningmethod is of great reproducibility, from one screen to another and theinteractions identified are thus of a high reliability.

[0023] In a specific embodiment of the method according to theinvention, which is a further improvement over the prior art methods,the DNA library is presented as a ready-to-use biological materialconsisting in a collection of recombinant haploid yeast cells containingthe whole inserts generated during the construction of the DNA libraryunder the form of prey polynucleotides as defined above, said collectionof yeast cells being frozen in multiple vials, each vial containing anidentical biological material.

[0024] Consequently, one vial is thawed for each screening experimentand is directly used in the cell-to-cell mating step, in contrast toprior art methods, for example as described by Bendixen et al. (1994),that need a first step of separate culture, in suitable selectivemedium, both of the recombinant yeast cell clones containing the baitpolynucleotide and of the recombinant yeast cell clones containing thechosen prey polynucleotides, then a second step of clone-to-clonereplica-plating which is also performed in rich culture medium, beforeanother step of clone-to-clone replica-plating for selecting therecombinant diploid yeast cells contained in the mated clones in aselection culture medium.

[0025] The above described characteristics of this specific embodimentof the screening method according to the invention ensures that saidmethod is fast, exhaustive and reproducible, in contrast with the priorart techniques.

[0026] The two-hybrid screening method according to the invention is farmore both quantitatively and qualitatively reproducible than the priorart methods that include a step of transformation of yeast cell withplasmidic DNA, for example, DNA originating from inserts contained in aDNA library prepared in E. coli.

[0027] Indeed, a primary DNA library in E. coli only allows a reducednumber of successive screenings without the need of a further culture ofthe E. coli clones in order to make more starting DNA materialavailable. In these circumstances, the further culture of therecombinant E. coli clones of the DNA library necessarily introducediscrepancies in terms of the representativity of the different insertsinitially contained in the DNA library, as the different clones may havevarious growth rates.

[0028] In contrast, the method according to the invention allows in asingle step the preparation of a high quantity of starting DNA materialunder the form of recombinant haploid yeast cells, said startingmaterial being subsequently stored in a high number of identical vials,thus ensuring that each processing of the method use strictly identicalstarting material representing the whole DNA library initially prepared.

[0029] The great reproducibility and exhaustivity of the above-describedmethod allows the one skilled in the art to reiterate said method usingeach of the prey polynucleotide selected at step b) as a bait in orderto identify and characterize polynucleotides that are systematicallyselected as coding for interactor polypeptides of biologicalsignificance.

[0030] The successive reiterations of the screening allow the oneskilled in the art to identify important interactions betweenpolypeptides encoded by diverse polynucleotide inserts contained in theinitial DNA library, which interactions are of statistical andbiological significance. Three reiterations of steps a) to c) of themethod according to the invention allow the one skilled in the art todetermine which polynucleotide inserts of the initial DNA library aresystematically selected for interaction with an initial bait polypeptideand/or another polynucleotide insert of the initial DNA library and thuswhich is statistically of great metabolical and/or physiologicalinterest.

[0031] Consequently, a specific embodiment of the method according tothe invention comprises repeating at least once steps a) to c) using,for performing each reiteration, at least one previously selected and/orcharacterized prey polynucleotide as the bait polynucleotide.

[0032] The number of repeats of steps a) to c) is no more than 10,preferably no more than 5 and in a most preferred embodiment the numberof reiterations of steps a) to c) is 3.

[0033] In a preferred embodiment of the method of the invention in whichsteps a) to c) are reiterated, the bait polynucleotide used thatcorresponds to a selected prey belongs to the following group ofpolynucleotide consisting in:

[0034] a) a polynucleotide that is identical to said selected preypolynucleotide;

[0035] b) a polynucleotide containing the complete ORF including saidselected prey polynucleotide;

[0036] c) a polynucleotide which is any polynucleotide fragmentcomprised in the complete ORF including said selected preypolynucleotide.

[0037] A polynucleotide fragment of a complete ORF may be obtainedeither by digestion with a restriction endonuclease, as described inSambrook et al., or by digestion with an exonuclease such as Bal1, oralso by DNA synthesis, such as described by Sonveaux et al. (1986),Hsiung et al. (1980), Froehler et al. (1986), Alvarado-Urbina (1986),Crea et al. (1978) or also Urdea et al. (1983) or by PCR as described inthe Examples.

[0038] Using, as the bait polynucleotide, a complete ORF correspondingto a given prey selected at a given round, for example first round, ofthe method of the invention, for performing the next round allows theone skilled in the art to select exhaustively all the potentialpolypeptides that are able to interact with the translation product ofsaid complete ORF bait polynucleotide. Consequently, all the possiblepolypeptides interacting with any peptide domain of the polypeptideencoded by the complete ORF including the previously selected prey areidentified.

[0039] Preferably, the first screening using the method according to theinvention will comprise a limited number of screenings with a limitednumber of bait polynucleotides, usually with bait polynucleotidesencoding for bait polypeptides already characterized for being involvedin a given physiological and/or metabolic pathway, like, for example,pre-mRNA splicing.

[0040] When several, preferably three, reiterations of the method of theinvention are performed and thus common bait and prey polypeptide areselected, a map of all the interactions between these polypeptides maybe designed, that take into account of the known and/or suspectedbiological function of each of the polypeptide interactor molecules.Such an interactors map may help the one skilled in the art to deciphera whole metabolical and/or physiological pathway that is functionallyactive within the host organism from which the initial DNA library isderived, as it will be seen in the examples presented hereunder.

[0041] Another object of the present invention consists in arepresentative and exhaustive genomic DNA library of an prokaryotic oran eukaryotic host organism that is prepared according to the invention.

[0042] Preferably, the method of the invention is performed using, asthe genomic DNA library starting material, inserts provided by thefragmentation of the genome of a host organism that does not contain,and/or contains a small number of, intronic sequences.

[0043] Preferably, such an exhaustive genomic DNA library is preparedfrom the genomic DNA of a host organism endowed with a compact genome,that is to say a genome containing at least 50% of coding sequences,more preferably at least 65% of coding sequences and most preferably 75%of coding sequences. Among such an host organism having a compact genomeas defined herein above may be cited prokaryotic organisms like virusand bacteria and also eukaryotic organisms such as yeast.

[0044] A further object of the present invention consists in arepresentative and exhaustive genomic DNA library derived fromSaccharomyces cerevisiae, designated as the FRYL library, which is usedwhen performing the two-hybrid screening method of the invention.

[0045] The invention also concerns an improved recombinant plasmid usedto express the prey polynucleotides to be selected according to themethod of the invention, as well as a recombinant host organismcontaining said plasmid.

[0046] The invention is also directed to a collection of recombinantcell clones consisting in a collection of recombinant host organisms asdescribed hereinabove.

[0047] The present invention concerns also a recombinant diploid yeastcell selected by the method of the invention.

[0048] Are also part of the invention a polynucleotide that has beenselected according to the method of the invention, as well as apolypeptide which is encoded by such a polynucleotide.

[0049] The invention is further directed to a technical mediumcontaining an interactors map representing at least a set of interactionevents that have taken place between the polypeptides encoded by thepolynucleotides selected by the method of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050]FIG. 1. Classification of preys selected in a two-hybrid screen ofa yeast genomic library: A fragment of a yeast chromosome is representedwith a potential in frame non sense codon (u) upstream of the initiationcodon of a yeast ORF (open box). The putative interacting domain isshown (shaded box). Arrows indicate the position, the length and theorientation of selected inserts. The categories of preys are defined onthe right (see text for details).

[0051]FIG. 2. A mating strategy for multiple round two-hybrid screens:See Experimental procedures for details.

[0052]FIG. 3. Western blot analyses of Gal4 fusion proteins used asbaits: Extracts of CG1945 cells transformed with the various baitplasmids were separated by a 12% SDS-denaturating gel, blotted andanalyzed with an anti-Gal4 DNA binding domain antibody. The expectedmolecular weight (kD) of the fision proteins is given in brackets. 1:pAS2ΔΔ (16); 2: Prp9p (79); 3: Prp11p (46); 4: Prp21p (49); 5: Cus1p(66); 6: Yor319w (40); 7: Yjr022w (30); 8: Yer029c (38); 9: Mud1p (50);10: Snp1p (50); 11: Mud2p (76); 12: Ylr116w (69); 13: Yir009w (29); 14:Ylr456w (40); 15: Smd1p (32); 16: Smd3p (27).

[0053]FIG. 4. Distribution profiles of preys found in two-hybridscreens: All preys selected with a bait indicated below each bar arerepresented according to the classification given in FIG. 1 (see insert;for clarity, A2 and A3 preys are grouped). Subdivisions within acategory reflect the number of preys falling within the same genomiclocus.

[0054]FIG. 5. Interacting domains defined by ORF fragments selected intwo-hybrid screens: FIG. 5A. The position of inserts selected in Mud2pand Ylr116w screens and covering Smy2p (top) and Lpg4p (bottom)sequences are shown. The numbers indicate the amino acid residues. Outof frame fusions are labeled with an asterisk with the −1 or the +1frameshift indicated. The minimal interacting domain is shaded. FIG. 5B.Inserts covering the Ylr116w sequence and selected in the Mud2p screenare shown. The alignment with the human SF1 protein is depicted with thepositions of the regions of high homology (hatched) and of the KR andKH-associated motifs (in black).

[0055]FIG. 6. The family of yeast Sm proteins: Yeast sequences aregrouped in sub-families and aligned with the respective humanhomologues. Dashes indicate omitted residues for sake of space. Residuesthat define Sm1 and Sm2 motifs are indicated by vertical grey bars(Hermann et al., 1995). Common residues within a family are shown inreverse print.

[0056]FIG. 7. Map of plasmid pAS2ΔΔ.

[0057]FIG. 8. Map of plasmid pACTIIst.

[0058]FIG. 9: A yeast protein interaction map: Labels are given in theinsert. Baits are boxed in black and white. All ORF preys found inscreens are shown and linked to their bait by an arrow. Preys of the A1category are boxed in black, and those of the A2 or A3 categories areboxed in grey. The unknown ORFs (square boxes) are named following theofficial nomenclature and the names of the known genes (rounded boxes)are those chosen by the Yeast Proteome Database. Smy2p and Ypl105c aretwo highly homologous proteins. Splicing factors selected as preys areunderlined. Preys found in several screens are separately boxed exceptSwi1p for which interactions are most probably non specific.

DETAILED DESCRIPTION OF THE INVENTION

[0059] The inventors have developed new tools and procedures for thetwo-hybrid strategy that allows a highly selective screening of acomplete genome, typically a compact genome such as the yeast genome,leading to a limited set of proteins potentially interacting with agiven protein.

[0060] Thus, the present invention is directed to an improved two-hybridscreening method comprising steps a) to c) described hereinbefore, and,as a specific embodiment of said method, at least one reiteration ofsteps a) to c) using at least one initial prey polynucleotide as thebait polynucleotide.

[0061] In a preferred embodiment of the method according to theinvention, the mating step between the two categories of recombinanthaploid yeast cells, respectively containing a given bait polynucleotideor a prey polynucleotide included in a collection of preypolynucleotides, is performed on a porous filter, the mean diameter ofthe pores being such that said filter retains the yeast cells.

[0062] The filter may be used as a component of a filter holder in whicha yeast cell mixture consisting in both a first population of haploidyeast cells containing the bait polynucleotide and a second populationof haploid yeast cells which is a collection of yeast cells, eachcontaining a different prey polynucleotide. In this specific embodimentof the method according to the invention, the filter holder is filledwith a culture medium suspension containing the yeast cell mixture whichis passed through the filter in order to collect the yeast cell mixtureon said filter before placing the filter on a suitable culture dishpreviously coated with a desirable culture medium.

[0063] The filter may also be placed directly on the culture dishpreviously coated with the desirable culture medium. In this case, theyeast cell mixture is added onto the already placed filter, in a smallvolume of culture medium, after that the initial yeast cell mixture hasbeen centrifuged and then harvested in said small volume of culturemedium, before plating, optionally using glass beads, the resultingmixture on a suitable culture dish previously coated with a desirableculture medium.

[0064] The mating step designed by the present inventors may beperformed in a short period of time such that practically nomultiplication of recombinant yeast cells in presence occur. Typically,the mating step has a duration of less than five hours and is preferablyperformed in a period of time of four hours or even less, provided thatthis period of time is at least three hours.

[0065] As already mentioned, the two-hybrid method of the inventionmakes use of at least one detectable gene, the transcription of which isactivated when a prey polypeptide and the bait polypeptide produced bythe diploid recombinant yeast cells interact, due to the triggering ofthe transcription of said at least one detectable gene when both theDNA-binding domain contained in the bait polypeptide and thetranscriptional activation domain contained in one prey polypeptide arein proximity, one to the other.

[0066] By at least one detectable gene according to the invention, it isintended from one to five, and preferably two or three detectable genes,the transcription of which is activated within the recombinant diploidyeast cell when the encoded bait and prey polynucleotides are able tointeract.

[0067] Introducing a redundancy in the detectable signals produced whenthe bait polynucleotide and a particular prey polynucleotide interactallows the one skilled in the art to discard the eventual artefacts thatcorrespond to background levels of transcription of one of thedetectable genes used in a given situation and consequently avoid theselection of false positive interaction and then false interactorpolypeptides or polynucleotides.

[0068] Preferably, the at least one detectable gene is contained by thefirst recombinant haploid yeast cell containing the bait polynucleotide.

[0069] The at least one detectable gene may be contained either in aplasmid of the recombinant diploid yeast cell or in its genome.

[0070] As an illustrative embodiment, the at least one detectable geneis located in the chromosome of one recombinant haploid yeast cell usedaccording to the method of the invention, and preferably the yeast cellcontaining the bait polynucleotide.

[0071] As another illustrative embodiment, the at least one detectablegene is choosen among the group consisting in a nutritional gene or alsoa gene the expression of which is visualized by colorimetry, such asHIS3, LacZ or both LacZ and HIS3.

[0072] The DNA-binding domain of the bait polypeptide and thetranscriptional activating domain of the prey polypeptide may be ofdifferent kinds. As an illustrative embodiment, these can be derivedfrom LexA or also Gal4.

[0073] Moreover, the inventors have also modified a prior art plasmid,plasmid pAS2 (commercialized by Clontech) in order to improve thefunctional characteristics of the plasmid containing the baitpolynucleotide by deleting two genes:

[0074] i) a first gene initially used as a selection marker, gene CYH2coding for the cycloheximide resistance and which have been found by theinventors to be of significant toxicity for the recombinant host cellsbearing it;

[0075] ii) a second gene, gene HA used for detecting the synthesis ofthe fusion polypeptides in western blotting experiments (using anti-HAantibodies), the presence of which has been found to cause a reducedselectivity.

[0076] The resulting improved plasmid is plasmid pAS2ΔΔ (See FIG. 7)which is contained in the E. coli strain that has been deposited at theCollection Nationale de Cultures de Microorganismes (C.N.C.M.) on Feb.,XX 1998 under the accession number I-XXXX. Plasmid pAS2ΔΔ is also partof the invention, as well as a recombinant organism of prokaryotic oreukaryotic origin containing said plasmid.

[0077] Is also part of the present invention the plasmid pACTIIst, therestriction map of which is presented in FIG. 8.

[0078] The invention also consists in a recombinant host organismcontaining the recombinant plasmid pACTIIst.

[0079] Is also part of the present invention a recombinant host organismcontaining the recombinant plasmid pAS2ΔΔ.

[0080] More specifically, the following recombinant haploid yeast cellsare useful for performing the method according to the present invention:

[0081] Y187, (MATα Gal4ΔGal80Δ ade2-101 his3 leu2-3,112 trp1-901 ura3-52URA3::GAL1_(UAS)-LacZ met⁻),

[0082] CG1945 (MATa Gal4-542 Gal80-538 ade2-101 his3Δ200 leu2-3,-112trp1-901 ura3-52 lys2-801 URA3::GAL4_(17mers(X3))-CyC1_(TATA)-LacZLYS2::GAL1_(UAS)-GAL1_(TATA)-HIS3 CYH^(R)),

[0083] L40 (MATa ade2 trp1-901 leu2-3,112 lys2-801am his3Δ200LYS2::(lexAop)₄-HIS3 URA3::(lexAop)₈-lacZ) (Hollenberg et al., 1995),and

[0084] L40ΔGAL4 (MTAa ade2 trp1-901 leu2-3,112 lys2-801am his3Δ200LYS2::(lexAop)₄-HIS3 URA3::(lexAop)₈-LacZ GAL4::KanMX2).

[0085] The L40ΔGAL4 yeast strain allows the one skilled in the art toselect the recombinant diploid yeast cells obtained according to themethod of the invention both with the HIS3 and the LacZ marker genes.The L40ΔGAL4 strain has been prepared according to the teachings of Wachet al. (1994, Yeast, 10:1793-1808).

[0086] Another object of the invention consists in a collection ofrecombinant cell clones from a host organism as described above, eachdifferent cell clone containing an inserted polynucleotide from a DNAlibrary. Preferred recombinant host organisms are E. coli and yeast suchas Saccharomyces cerevisiae.

[0087] An important feature of a further specific embodiment of thepresent invention resides in that the initial DNA library which providesthe prey polynucleotides is strictly representative of the genome fromwhich this DNA library is derived. By strictly representative, it isintended that the initial DNA library is composed of a number ofpolynucleotide inserts such that the DNA library i) covers the wholegenome of interest and ii) the 5′ end of the polynucleotide insertsbegins every at least four bases interval of the genome of interest.

[0088] As a general feature of the method according to the presentinvention, the prey polynucleotides to be selected are provided by a DNAlibrary. Such a DNA library may be prepared either from cDNA or alsogenomic DNA previously fragmented by restriction enzymes or sonication.For the use of restriction enzymes, the one skill in the art may referto the works of Sambrook et al. (1989).

[0089] In one specific illustration, said DNA library is prepared using, as starting material, the mRNA or the genome of a prokaryotic hostorganism.

[0090] In another specific embodiment of such a DNA library, thestarting material is the mRNA or the genome of a eukaryotic hostorganism.

[0091] A large and extremely representative genomic DNA library of aprokaryotic or eukaryotic organism having a compact genome is preparedby sonication as described in the Materials and Methods Section. Oneparticular characteristic of such a DNA library is that all thepotential interactors naturally encoded by the source organism arerepresented. Another characteristic of said DNA library consists in thatno misrepresentation (i.e. over- or under-representation) of specificcoding sequences are observed, as regards to the initial source genome.

[0092] Such a DNA library is prepared by sonication of the sourcegenomic DNA, ensuring a random cleavage of the starting DNA material andthus a excellent representation of all the possible inserts, in contrastto techniques using restriction endonucleases.

[0093] In the protocol described, a great care have been taken inperforming the steps of ligation of the DNA fragments that have beengenerated by the sonication step.

[0094] As an illustrative embodiment of such an exhaustive andrepresentative DNA library used when performing the method according tothe invention, the whole genome of Saccharomyces cerevisiae has beenrandomly fragmented by sonication and the resulting DNA fragments havebeen cloned in order to build a genomic DNA library. Said genomic DNAlibrary contains 3.6×10⁶ different clones derived from the Saccharomycescerevisiae genome that comprises about 15×10⁶ nucleotides. In otherwords, the Saccharomyces cerevisiae DNA library realized by theinventors contain polynucleotides beginning at every four bases intervalof the initial genome.

[0095] As an illustration of the present invention, when applied to anexhaustive and representative genomic library of the yeast, and morespecifically of Saccharomyces cerevisiae, several known proteinsinvolved in the same pathway are used as baits in a first round oftwo-hybrid screens. Among the produced sets of preys, a new series ofbaits is in turn used for second round screens. Repeating this procedureseveral times have lead to the characterization of a network ofinteractions. Therefore, starting from known proteins, this approach notonly makes connections between known proteins, but also identifies newfactors and suggests novel functional links between pathways so farunrelated.

[0096] The spliceosome is an attractive entry point for such a project:i) the spliceosome formation is a dynamic process conserved throughevolution that requires RNA-RNA interactions, protein-RNA interactionsand protein-protein interactions; ii) it is formed of several snRNPparticles, each of them containing many different proteins; iii) itbuilds on pre-mRNA through a stepwise formation of different complexes;iv) pre-mRNA splicing is a cellular process that is functionally linkedto transcription and processing of primary transcripts, including exportto the cytoplasm. Altogether, the implication of numerous differentproteins suggests a pivotal role of the protein-protein interactions notonly within each snRNP but also between particles. Moreover, the linkbetween pre-mRNA splicing and other nuclear processes is most likelymediated by protein-protein interactions.

[0097] Spliceosome assembly involves the U1, U2, U4/U6 and U5 snRNPs andmany additional splicing factors that were characterized through geneticscreens and in vitro biochemical analyses (Beggs, 1995). Sequenceanalyses indicate that most of the yeast factors have homologues inhuman cells (Kr{haeck over (S)}mer, 1996). Out of several dozen yeastsplicing factors, we chose some which are implicated in early steps ofspliceosome formation.

[0098] A new yeast genomic library has been constructed and improved thetwo-hybrid procedure in such a way that for each bait, the completescreening of the genome produced a very limited set of candidates. Fivedifferent categories of candidates have been defined. Among thecandidates, several were chosen for subsequent two-hybrid screens, and anovel prey was again used as bait in a third round of screening. On thewhole, the fifteen screens led to the characterization of newinteractions between known splicing factors, to the identification ofnew yeast splicing factors homologous to already known mammalian factorsand to the finding of unexpected interactions that open novelopportunities for functional analyses.

[0099] As already mentioned hereinbefore, the inventors have constructeda novel and improved genomic DNA library from Saccharomyces cerevisiaecontaining 5×10⁶ independent cell clones that have been pooled toconstitute the “FRYL library.” More than 70% of the clones testedcontained an insert the nucleotide length of which is in the range from200 to 1400 nucleotides with an average length of 700 bases.

[0100] The characteristics of the FRYL library allows the one skilled inthe art to design DNA constructions for use in the method according tothe invention leading to an in frame fusion between the Gal4 activationdomain and a yeast ORF once every 24 nucleotides.

[0101] When assaying the fusions created using the polynucleotideinserts of the FRYL library as starting material, and using, on anotherhand, bait polynucleotides encoding already known nuclear proteinsimplicated in the pre-mRNA splicing pathway (listed in Table 1) severalpolynucleotide preys originating from the FRYL library were selectedwhich have been determined to be of great biological significance, sinceamong the selected prey polynucleotides a significant number of thoseare involved in the pre-mRNA splicing pathway, including stilluncharacterized ORFs (Ylr116w and Yor319w) sharing strong homology withhuman protein counterparts such as SAP49 and SF1, one of the human SF3bcomponent (Wells et al., 1996; Arning et al., 1996).

[0102] Thus, is also part of the present invention the FRYL librarycontained in the collection of recombinant E. coli strain MR32 cellsthat have been deposited at the Collection Nationale de Culture deMicroorganismes (C.N.C.M.) on Dec. 21, 1995 under the access numberI-1651.

[0103] The present invention also concerns a recombinant diploid yeastcell selected by the present two-hybrid method.

[0104] Another object of the invention consists in a polynucleotide thathas been selected with the method of the invention, as well as apolypeptide that is encoded, at least in part, by said polynucleotide.

[0105] The invention is also directed to technical means that are neededto perform the present two-hybrid screening method.

[0106] An illustration of such technical means consists in a kit forselecting at least one polypeptide of interest belonging toSaccharomyces cerevisiae, wherein said kit comprises

[0107] a) at least one complete collection of yeast cell clonescontaining the whole polynucleotide inserts representative of the genomefrom a prokaryotic or eukaryotic organism having a compact genome;

[0108] b) optionally, the plasmid pAS2ΔΔ;

[0109] c) optionally, the plasmid pACTIIst;

[0110] d) optionally, a haploid yeast cell to be transformed with aplasmid containing a bait polynucleotide of interest;

[0111] e) optionally, the reagents necessary to visualize the expressionof at least one detectable gene, such as X-Gal.

[0112] The most preferred embodiment of a genomic DNA library usable asa starting material in a kit according to the invention is the FRYLlibrary described above, which may be initially contained in plasmidsthat have been transformed in E. coli (CNCM I-1651), or which may bedirectly contained in a suitable recombinant haploid yeast cell (FRYLlibraries to be deposited).

[0113] As it is described in details in Example 4, a second and thirdreiteration of the method according to the invention, using thepreviously selected prey polynucleotides as the bait polynucleotidesthat are again tested against the FRYL DNA library, have allowed theinventors to design a map of the identified interactions (See FIG. 9).More precisely, as it can be seen in FIG. 9, 170 preys have beenselected in the 15 screens performed and correspond to 145 ORFs,implicated in different cellular pathways. Nine Orfs are involved inpre-mRNA splicing, six in other RNA metabolisms, ten are involved intranscription and four are protein kinases and phosphatases.

[0114] Since the screening method of the invention is exhaustive andreproducible, the selected prey polynucleotides may be classified indifferent categories, depending of their degree of occurrence in thepositively selected diploid yeast cell clones or also of their sizelocation or their location in a given ORF.

[0115] Consequently, is also part of the invention a particularembodiment of the present two-hybrid method, wherein the preypolynucleotide selected at step b) is classified within one of thefollowing prey polynucleotide classes:

[0116] a) a polynucleotide contained in an intergenic region or on thereverse orientation of an ORF contained in the genome of the organismfrom which the initial DNA library has been prepared (B).

[0117] b) a polynucleotide selected several times and contained indifferent clones of the initial DNA library (A1);

[0118] c) a polynucleotide selected only once and having a 5′ end of thecoding strand starting close to an initiation codon of an ORF containedin the genome of the organism from which the initial DNA library hasbeen prepared (A-2);

[0119] d) a polynucleotide selected only once and having a nucleotidelength of at least 1000 bases (A3);

[0120] e) a polynucleotide selected only once and having characteristicsdifferent from the polynucleotides of the a), b), c) and d) classes(A4).

[0121] For the purpose of the present invention, the expression “codingstrand starting close to an initiation codon of an ORF” means that theinitiation codon of a given yeast ORF is at a nucleotide distance lessthan 150-200 bases from the in-frame stop codon located upstream saidORF.

[0122] Among the polynucleotides inserts selected by the methodaccording to the invention, several of them include inserts with out offrame fusions. The hypothesis is that the correct in-frame synthesisoccurs through the production of frameshifted polypeptides.

[0123] Since the two-hybrid screening method of the invention allows theone skilled in the art to design a statistically and biologicallysignificant map of the interactor polypeptides (baits and preys) thathave been selected, another object of the invention is a technicalmedium containing the whole pertinent interactions between metabolicallyrelated bait and prey polypeptides and/or polynucleotides coding forsuch bait and prey polypeptides.

[0124] As a specific embodiment of such a technical medium definedherein above, it may be cited a computer useable medium containingcomputer readable data related to the interactions between at least onebait polypeptide and at least one prey polypeptide encoded at least inpart by a prey polynucleotide that has been selected with the two-hybridscreening method of the invention.

[0125] The present invention will be fully illustrated by the Materialsand Methods protocols and by the Examples described below, although thescope of the invention cannot in anyway be limited to these specificembodiments.

[0126] Materials and Methods

[0127] A: The Mating Experiment

[0128] The mating procedure allows a direct selection on selectiveplates because the two fusion proteins are already produced in theparental cells. No replica plating is required. We routinely obtain atleast 20% mating efficiency and regularly 50% mating efficiency.

[0129] Usually, the Y187 strain is transformed either with the FRYLyeast genomic library (see Part J for the characteristics of the yeastgenomic FRYL library). Independently, bait plasmids are introduced inthe CG1945 strain. The Y187 strain contains a sensitive LacZ reportergene whereas the CG1945 strain has a non-leaky HIS3 reporter gene thatenables the selection of positive diploids in the absence of3-aminotriazol (3-AT).

[0130] Alternatively, bait plasmids can be introduced into the L40strain. This plasmid derived from pBMT116 plasmid. It encodes for LexAfusion protein that can be assayed in a two hybrid system in the L40strain containing His3 and LacZ reporter genes downstream LexA bindingsites. The same Gal4-derived libraries could be used. However, the L40strain is not deleted for the GAL4 gene and although glucose repressionoccurs, a residual activation of this gene in the diploid cells promotesthe transcription of the parental Y187 strain's LacZ reporter gene.Thus, in a L40×Y187 diploid cell, selection is made only on the HIS3reporter gene.

[0131] This protocol is written for the use of the FRYL yeast genomiclibrary cloned into the Y187 strain. Any other genomic or cDNA librarymight be used with minor adaptations to take into account theircomplexity and the cell density of the frozen cells.

[0132] Strains

[0133] CG1945 (MATa Gal4-542 Gal80-538 ade2-101 His3-200 Leu2-3,-112Trp1-901 Ura3-52 Lys2-801 URA3:: GAL4 17mers (X3)-CyC1TATA-LacZLYS2::GAL1UAS-GAL1TATA-HIS3 CYH^(R)) transformed with the pAS2ΔΔ baitplasmid (see FIG. 7 for plasmid map)

[0134] Y187 (MATα Gal4Δ Gal80Δ ade2-101 His3 Leu2-3,-112 Trp1-901Ura3-52 URA3::UASGAL1-LacZ Met⁻) transformed with the FRYL yeast genomicDNA library (see Part L).

[0135] Day 1 Preculture

[0136] Materials

[0137] 100 ml flask with 20 ml -W medium

[0138] Experiment

[0139] Preculture of CG1945 cells carrying the bait plasmid in 20 ml -Wmedium

[0140] Grow at 30° C., vigorous agitation.

[0141] Day 2 Culture

[0142] Materials

[0143] 1 liter flask with 150 ml -W medium

[0144] Experiment

[0145] at 6 pm

[0146] Estimate OD₆₀₀ of the -W preculture of CG1945 cells carrying thebait plasmid. Measure OD₆₀₀ of 4 to 10 fold dilution depending of thepreculture. The OD must lie between 0.1 and 0.5 in order to correspondto a linear measurement (1 OD=10⁷ CG1945 cells using ourspectrophotometer).

[0147] Inoculate 150 ml -W at OD₆₀₀ 0.006/ml (may depend on local growthconditions; to be tested with various dilutions).

[0148] Grow o.n. at 30° C., vigorous agitation.

[0149] Day 3 Mating

[0150] Materials

[0151] All the Material Must Be Sterile

[0152] Medium and Plates

[0153] 15 YPglu+Tetracyclin (Tet) 6 mg/ml plates

[0154] a 50 ml tube with about 50 ml YPglu+Tet (6 mg/l).

[0155] a 50 ml tube with 35 ml -LWH

[0156] 100 ml flask with 20 ml of YPglu

[0157] 75 -LWH+Tet (6 mg/ml) plates

[0158] 2 -L plates

[0159] 2-W plates

[0160] 2 -LW plates

[0161] Materials

[0162] Swinnex 47 filter holder (ref: millipore SX00 047 00)

[0163] filters 0.22 μ 45 mm (ref: millipore GSWP 047 00)

[0164] 10 ml syringe

[0165] 9 cm plates

[0166] forceps

[0167] multipipettor and 10 ml sterile tips

[0168] glass beads 3 mm

[0169] 50 ml tubes

[0170] large beaker

[0171] Experiment

[0172] A.M.

[0173] Rinse forceps with water and ethanol; flame it and place itinside a sterile 9 cm plate. Using the syringe, soak up 1 ml of YPgluand keep it also inside a sterile 9 cm plate. Mark the YPglu+Tet plateswith bait name.

[0174] Prepare Filter holder: open it up; (place parts inside a 9 cmplate) check placing of rubber rings; wet the filter-part with YPglu(from the syringe) and place a 47 mm filter on top, in the middle. Letthe filter soak up the liquid and screw the upper part of the filterunit tightly on the lower half. Rinse filter with about 10 ml YPgluapplied using the syringe attached to the opening at the top of thefilter holder. Collect the liquid in the 500 ml beaker. Press theplunger to get an even distribution of the liquid over the filter. Checkthat the filter unit doesn't leak. If it does, don't panic, take off thepressure, open the unit, replace the filter and/or the rings and screwit maybe more tightly.

[0175] If the filter unit is contaminated take a new one and start again(This can also happen during the collection of diploid cells asdescribed below as you replace the filter every time. Only when you takea new filter holder you have to do the pre-wetting and to rinse with 10ml of YPglu).

[0176] Measure OD₆₀₀ of the -W culture of CG1945 cells carrying the baitplasmid. It should be around 1; definitely not higher than 1.5.

[0177] For the mating you must use twice as many bait cells as librarycells. A vial of the Y187/FRYL yeast genomic DNA library contains 4.10⁸viable cells.

[0178] Estimate the amount of bait culture (in ml) that makes up 80OD₆₀₀ units for the mating with the yeast library (1 OD=10⁷ CG1945cells).

[0179] Thaw a vial containing the Y187/library slowly on ice.

[0180] Add the contents of the vial to 20 ml YPglu using a sterileplugged 1 ml pipet; rinse the vial with culture liquid.

[0181] Let those cells recover at 30° C., under gentle agitation for 10minutes. Set your timer.

[0182] Mating

[0183] Put 80 OD₆₀₀ units of bait culture into a 250 ml flask. (Do notleave the CG1945 cells without agitation because of the aggregation ofthe cells)

[0184] Add the Y187/library culture to the bait culture.

[0185] Mix the library/bait cells by hand.

[0186] Collect 1/15 volume of mixed library/bait culture on one filterby pressing it through the Swinnex filter holder (15 independent matingexperiments will be done). Rinse the filter with 4 ml fresh YPglu andabout 10 ml air to dry it. Take off the syringe (under-pressure mightcause a suck back; try to prevent it). Repeat the pressure with 10 mlair until no medium goes through the swinnex. Open the filter unitcarefully so that any remaining fluid stays on top of the filter. Usingthe forceps, take off the filter and place it cells-up on top of anYPglu+Tet plate.

[0187] Repeat this another 14 times until all the cells are collected onfilters. Before every sampling shake the culture to counteract theflocculation and to homogenize the cell density.

[0188] Incubate plates cells-up at 30° C. for 4 h 30 mn.

[0189] P.M.

[0190] Collection of Mated Cells

[0191] Mark the -L, -W, -LW and -LWH+Tet plates with date and bait-name.Add 5 to 7 sterile glass beads to every plate.

[0192] Wash and flame forceps (before placing them inside a sterile 9 cmplate).

[0193] Place the base of a 9 cm plate against the base of a Bunsenburner. Place a filter, with mated cells up, inside the base. Rinse thecells from the filter with 1 ml of -LWH using a pipetman. Reapply thecell-slurry a couple of times until all cells are washed off. Pipet thecell-slurry into the collection tube. (The cells are clearly visible asa reddish layer and come off as sheets in the beginning). Wash andrerinse the filter with another ml -LWH and pipet this into thecollection tube. Discard filter with original YPglu+Tet plate.

[0194] Repeat this with all the filters. You will end up with about 30ml of cell-suspension. Take note of the actual volume.

[0195] Plate the controls

[0196] Shake cell-suspension and make a 1:1000 dilution by three 10 folddilution steps: (50 μl mix to 450 μl of fresh -LWH medium, shake well).Spread 50 μl of the 1000 fold dilution on -L, -W and -LW plates.Incubate cells down at 30° C. for two days.

[0197] Plate the Screen

[0198] Take multipipettor and sterile 10 ml tips.

[0199] Shake cell suspension and distribute cells in 400 μl samples overthe 75 -LWH+Tet plates with glass beads. Spread cells by shaking theplates. Incubate plates cells down at 30° C. for three days.

[0200] This can be done only if one knows the behavior of the bait (e.g.it does not require addition of 3-AT in the plates). Otherwise, for anunknown bait, the optimal conditions must first be determined by platingsamples onto -LWH plates without and with 3-AT at various concentrations(ranging from 1 to 50 mM).

[0201] The cell mixture can be stored at 4° C. and plated 3 days afterthe mating.

[0202] Day 3 Mating (Alternative Protocol)

[0203] Materials

[0204] All the Material Must Be Sterile

[0205] Medium and Plates

[0206] 5 YPglu+Tetracyclin (Tet) 6 mg/ml plates

[0207] a 50 ml tube with 30 ml -LWH

[0208] 100 ml flask with 20 ml of YPglu

[0209] 75 -LWH+Tet (6 mg/ml) plates

[0210] 2 -L plates

[0211] 2 -W plates

[0212] 2 -LW plates

[0213] Materials

[0214] 5 Hybond C-extra filters (Amersham, RPN 82 E)

[0215] 9 cm plates

[0216] forceps

[0217] multipipettor and 10 ml sterile tips

[0218] glass beads 3 mm

[0219] 50 ml tubes

[0220] Experiment

[0221] A.M.

[0222] Rinse forceps with water and ethanol; flame it and place itinside a sterile 9 cm plate. Mark the YPglu+Tet plates with bait name.Using the forceps, place a Amersham filter on top of an YPglu+Tet plate.Add 5 to 7 sterile glass beads to every plate. Prepare 5 plates withfilter and glass bead.

[0223] Measure OD₆₀₀ of the -W culture of CG1945 cells carrying the baitplasmid. It should be around 1; definitely not higher than 1.5.

[0224] For the mating you must use twice as many bait cells as librarycells. A vial of the Y187/FRYL yeast genomic DNA library contains 4.10⁸viable cells. To get a good mating efficiency you must collected thecells at 4.5.10⁶ cells per cm² of filter.

[0225] Estimate the amount of bait culture (in ml) that makes up 80OD₆₀₀ units for the mating with the yeast library (1 OD=10⁷ CG1945cells).

[0226] Thaw a vial containing the Y187/library slowly on ice.

[0227] Add the contents of the vial to 20 ml YPglu using a sterileplugged 1 ml pipet; rinse the vial with culture liquid.

[0228] Let those cells recover at 30° C., under gentle agitation for 10minutes. Set your timer.

[0229] Mating

[0230] Put 80 OD₆₀₀ units of bait culture into a 250 ml flask. (Do notleave the CG1945 cells without agitation because of the aggregation ofthe cells).

[0231] Add the Y187/library culture to the bait culture. Mix thelibrary/bait cells by hand. Transfer the mixture of diploids into 50 mlsterile tubes. Centrifuge the cells at 5000 g (4000 rpm) for 3 min.

[0232] Discard the Supernatant

[0233] Resuspend the pellet with 2 ml of YPGlu medium.

[0234] Distribute cells in 400 μl samples over the filter on YPGluplates with glass beads. Spread cells by shaking the plates.

[0235] Incubate plates cells-up at 30° C. for 4 h 30 mn.

[0236] P.M.

[0237] Collection of Mated Cells

[0238] Mark the -L, -W, -LW and -LWH+Tet plates with date and bait-name.Add 5 to 7 sterile glass beads to every plate.

[0239] Wash and flame forceps (before placing them inside a sterile 9 cmplate).

[0240] Place the base of a 9 cm plate against the base of a bunsenburner. Place a filter, with mated cells up, inside the base. Rinse thecells from the filter with 4 ml of -LWH using a pipetman. Reapply thecell-slurry a couple of times until all cells are washed off. Pipet thecell-slurry into the collection tube. (The cells are clearly visible asa reddish layer and come off as sheets in the beginning). Wash andrerinse the filter with 2 other ml -LWH and pipet this into thecollection tube.

[0241] Discard Filter with Original YPglu+Tet Plate

[0242] Repeat this with all the filters. You will end up with about 30ml of cell-suspension. Take note of the actual volume.

[0243] Day 5 Estimation of Diploid Number

[0244] Late in the afternoon: check controls of mating as well as someselective -LWH plates. About a hundred diploid colonies should bevisible on -LW plates. Multiplying this number by 20 (50 μl sample),1000 (dilution factor) and 30 (volume in ml of the diploids mixture)gives the amount of diploids (i.e. 60.10⁶ when you counted 100 on the-LW plate: 100×20×30×1000).

[0245] Count colonies on control plates. Estimate the mating efficiencyby dividing the number of colonies on -LW plate by the number on the -Lplate and multiply by 100.

[0246] Colonies should become visible on -LWH+Tet plates.

[0247] B: X-gal-Overlay Assay

[0248] Introduction

[0249] X-Gal-overlay assay is performed directly on the selective mediumplates after scoring the number of His⁻ colonies. This procedure is lesssensitive than the filter assay (Protocol C) but it is less timeconsuming and has the advantage of a better recovery of cells since itdoes not require freezing the cells in liquid nitrogen.

[0250] Materials

[0251] Work under the hood. Dimethylformamide (DMF) is toxic.

[0252] Set up a waterbath. The water temperature should be 50° C.

[0253] Stock solutions:

[0254] 0.5 M Na₂HPO₄ pH7.5 (71 g Na₂HPO₄ per 1 liter+4 mlorthophosphoric acid). Distributed per 250 ml and stored at 50_(i)C(neither filtrated nor autoclaved).

[0255] 1.2% Bacto-agar distributed per 210 ml (2.52 g/210 ml).Autoclaved and stored at 50° C.

[0256] (Alternatively, phosphate and agar solutions are kept at roomtemperature and the solutions are mixed just before use after meltingthe agar in a microwave oven)

[0257] 2% X-Gal in DEF and stored at −20° C.

[0258] Overlay mixture:

[0259] 0.25 M Na₂HPO₄ pH 7.5

[0260] 0.5% agar

[0261] 0.1% SDS

[0262] 7% DMF (LABOSI ref DO675)

[0263] 0.04% X-Gal (ICN ref 150001)

[0264] For every plate you need 10 ml overlay mixture:

[0265] 5 ml 0.5M phosphate buffer pH 7.5

[0266] 4.2 ml 1.2% agar

[0267] 200 μl 2% (w/v) X-Gal in DMF

[0268] 100 μl 10% SDS

[0269] 500 μl DMF

[0270] -LWH plates

[0271] Sterile toothpicks

[0272] Experiment

[0273] Prepare the amount of overlay-mix you need. 500 ml maximum forone batch in order to keep the conditions similar (DMF will evaporate)and to prevent the mix settling too long in the bottle.

[0274] Mix 250 ml 0.5 M phosphate buffer pH 7.5+210 ml 1.2% agar+5 ml10% SDS. Go under the hood and add 25 ml DMF and 10 ml 2% X-Gal.

[0275] Don't add X-gal solution unless you will use it immediately.Temperature of the mix should be between 50° C. and 45° C. Highertemperatures might affect the cells; lower temperature impairs handling.

[0276] With pipetpump and sterile 25 ml pipet divide the overlay-mixover the plates in portions of 10 ml. Let the mix flow out graduallywhere there are no colonies growing. Too fast a flow or directly hittinga colony will cause smearing of the yeast cells. Too slow a flow willcause settling of the mix before it has spread equally over the plate.

[0277] Work through the whole batch of plates laid out. Collect themwhen the top layer is settled (keep the plates horizontal; don't tipthem; the whole overlay layer might move, causing smearing of thecolonies). Check whether all colonies are covered by the overlay. Ifnot, apply some mix on the ‘uncovered’ spots.

[0278] Incubate plates cells/overlay-up at 30° C. Note the time.

[0279] Check for blue colonies after 30 min, 1 h, 2 h, 4 h and 6 hincubation time. Mark positives with a felt-tip pen (a differentcolor/marking-style for each check at the different incubation time).

[0280] After 30′ and 1 hour do a quick check for blue colonies. Checkvery superficially. Do more careful checking after 2, 4 and 6 hours.

[0281] During checking, take the plates to the hood and check the coloragainst a black background. Don't inhale the DMF, keep your head out ofthe hood. At this moment it is not essential whether you are able topick all blue colonies (some will be pale).

[0282] Number the positives (If you want you can sample them in classes,distinguishing them according to colony-size and intensity ofblue-color).

[0283] Take some fresh -LWH plates (it is important to maintain theselection for the interaction on −LWH plates) and divide into 4sections. Number them according to the number of positives found. Markthem with date and bait-name.

[0284] Streak the positive colonies after 6 h incubation time. Put therest back at 30° C.

[0285] To streak cells from a single positive colony take a steriletoothpick through the agar-overlay into the colony and collect somecells. Streak the cells into one line. Take a fresh toothpick andrestreak cells from the first line into a second one. Take a thirdtoothpick and streak cells from the second into a third line and, afterturning the toothpick, into a fourth line.

[0286] Next day, check plates for positives. Streak newly foundpositives. It is important not to wait too long in doing this as thecomponents in the overlay-mix are not very good for the yeast cells(After 24 h incubation time the cells might be dead).

[0287] It takes about two days for colonies to grow.

[0288] There are five items to keep in mind:

[0289] 1) What counts as a ‘real’ positive (=blue color) can differ fromone screen to another. It might be related to the nature of the baitfusion, and also to its expression level.

[0290] 2) Another factor is how fast the yeast colonies arepermeabilized. Therefore you will see blue colonies coming up after anhour whereas for other colonies, it takes six hours. The intensity ofthe blue color can also range from deep dark to almost grey-light blue.Sometimes you see a blue halo around the colonies or the color is onlyrestricted to the colony or its center. Always compare the color ofputative positives with that of colonies on the same plate or on platesfrom the same screen.

[0291] 3) Because of these kind of differences keep track of the time atwhich positives are found, size of colony and intensity of blue-color.

[0292] 4) In the overlay method the blue-color develops with time.Compare the blue color of the positive colonies at a later time (i.e.after 24 h) than when you found/streaked them. Strong positives willturn bluer than weaker ones. Colonies that turned blue at a latertime-point than others might have developed a more intense blue colorthan the ‘earlier’ ones.

[0293] 5) Recovery of small colonies can be difficult, especially whenstreaking after 24 h incubation.

[0294] C: Filter Lift X-Gal Assay

[0295] Introduction

[0296] The filter lift assay is a fast and sensitive method to checkwhether the reisolated colonies are positive and homogeneous.

[0297] Materials

[0298] Plates with healthy, well-grown yeast colonies

[0299] Hybond C-extra filters (Amersham, RPN 82 E)

[0300] incubation plastic boxes

[0301] Whatman 3 MM paper

[0302] Millipore forceps

[0303] Saran wrap

[0304] Liquid Nitrogen

[0305] sterile toothpicks

[0306] -LWH plates

[0307] 2% (w/v) X-gal solution in Dimethylformamide (DMF)

[0308] 1M Na₂CO₃

[0309] Z-buffer with β-mercaptoethanol (Z-βOH):

[0310] 100 mM Na₂HPO₄ pH7.5

[0311] 10 mM KCl

[0312] 1 mM MgSO₄

[0313] add 1.8 ml β-mercaptoethanol per 500 ml Z buffer (50 mM) justbefore use (if added in advance keep solution at 4° C.).

[0314] To 20 ml Z-βOH buffer add 400 μl 2% X-gal solution.

[0315] Experiment

[0316] Take a sheet of Whatman paper upon which you can work.

[0317] Cut a sheet of Whatman that fits exactly into the bottom of theincubation box.

[0318] Prepare just enough Z buffer-βOH/Xgal mix for wetting the paper:For one incubation box (about 300 cm² of paper) you'll need about 20 mlZ-βOH/Xgal mix.

[0319] Pour the Z-βOH/Xgal mix over the paper. Start in the middle andtilt the box so that the paper is uniformly wetted. Get rid of anybubbles. (Try to lift the wet paper with forceps at one of the cornersto let the bubbles escape; be careful the paper may tear). Pour offexcess Z-βOH/Xgal mix and keep as back-up. Place the lid on the box.

[0320] Take a Hybond C-Extra filter (or enough to cover thoseplate-sectors where yeast colonies have formed), remove protectingpapers. Take a ball-point and mark the filter with a letter/number and aline.

[0321] Mark the plates with a line and numbers corresponding to thefilters.

[0322] Lift the marked filter, with the mark at the underside (i.e.towards the yeast cells), using the forceps (or by hand with gloves) andthe lid of the yeast plate.

[0323] Position the filter above the yeast plate so that the lines onthe plate and the filter are aligned.

[0324] Gently let the filter descend upon the yeast cells (i.e. notsmearing the colonies). Tap the plate in order to enhance the wetting ofthe filter.

[0325] Close the plate with the lid and, in the case you have to do alot, apply a filter to the next plate.

[0326] When the filter is uniformly soaked or when all plates are done,lift the (first) filter gently from the plate using the forceps.Directly hang the filter in liquid nitrogen without releasing your gripon the forceps. Count to five, take the filter out of the nitrogen andlet thaw cells-up on your paper work-sheet. (Continue with the nextfilter). Repeat freezing/thawing step a second time.

[0327] When the filters have thawed, transfer them to the incubation box(containing the Z-§OH/Xgal pre-wetted Whatman filter). Lay filterscarefully cells-up and without trapping air bubbles. Close the box andseal the lid with parafilm. Incubate at 30° C.

[0328] Prepare a stop-paper and a wash-paper:

[0329] Stop-paper: Take a slightly smaller piece of Whatman paper andplace inside an incubation box. Wet the paper with 1M Na₂CO₃.

[0330] Wash-paper: idem, but wet the paper with distilled water.

[0331] After 3 h incubation time, transfer the filters from theincubation box on the stop-paper and let them sit for 1 min. Transferthem to the wash-paper for another min., then dry them on a fresh pieceof Whatman paper.

[0332] Store the filters by sticking them to a new sheet of Whatmanpaper using doublesided tape. Keep the filters that belong to one screentogether. Wrap the papers with saran-wrap and seal the edges. Storetogether the filters belonging to the same screen.

[0333] Restreak cells from a positive colony after checking by thefilter lift assay (for each clone). Take some fresh -LWH plates anddivide into 8 sections. Number them according to the number of positivesfound. Mark them with date and bait name. Restreak cells from a singlepositive colony with 3 sterile sticks as described above to get isolatedcolonies.

[0334] D: PCR on Yeast Colonies

[0335] Introduction

[0336] PCR amplification of fragment of plasmid DNA directly on yeastcolonies is an efficient procedure to identify sequences cloned intothis plasmid. It is directly derived from a published protocol (Wang.Het al, Analytical Biochemestry, 237, 145-146, 1996). However, it is nota standardized protocol: in our hands it varies from strain to strain,it is dependent on experimental conditions (number of cells, Taqpolymerase source, etc.). This protocol should be optimized to specificlocal conditions.

[0337] Materials

[0338] 10× PCR buffer:

[0339] 500 mM KCl

[0340] 100 mM Tris HCl pH 9.0

[0341] 15 mM MgCl₂

[0342] 10 mM dNTP (mixture of the four dNTP at 10 mM each)

[0343] Taq polymerase (5 U/μl) from Pharmacia (ref. 27.0799-02)

[0344] 1 N NaOH

[0345] oligonucleotide upstream 100 ng/μl (oligo 52 for the W strand ofthe pACTIIst prey plasmid):

[0346] 5′-CGC-GTT-TGG-AAT-CAC-TAC-AGG-GAT-G-3′

[0347] oligonucleotide downstream 100 ng/μl (oligo 53 for the C strandof the pACTIIst prey plasmid):

[0348] 5′-GAA-ATT-GAG-ATG-GTG-CAC-GAT-GCA-C-3′

[0349] (see FIG. 8 for the sequence of pACTIIst plasmid).

[0350] Experiment

[0351] Take one colony with a toothpick. Resuspend the cells at roomtemperature in 10 μl 0.02N NaOH in an Eppendorf tube by turning thetoothpick for several seconds in the NaOH solution. Prepare 0.02N NaOHjust before use (5 μl 1N NaOH+245 μl water).

[0352] Incubate 5 min. at 100° C. For a large series, vortex the tubesbefore the incubation at 100° C. to counteract the sedimentation of thecells.

[0353] Put on ice immediately. Centrifuge the tube briefly to spin downthe drops of condensation on the cap.

[0354] Vortex to resuspend the pellet and transfer 2 μl of the cellextract in a 0.5 ml PCR tube preincubated in an ice/water bath.

[0355] Prepare the PCR mix for X reactions (at least one more -or 10%more for large series-than the actual number of reactions).

[0356] For One Reaction

[0357] 24.6 μl H₂O (qsp 30 μl)

[0358] 3.2 μl 10× PCR buffer

[0359] 0.7 μl 10 μl dXTP

[0360] 0.5 μl oligo 52

[0361] 0.5 μl oligo 53

[0362] (0.5 μl Taq polymerase)

[0363] Mix all the components except the Taq enzyme.

[0364] Go to the thermal cycler. The block of the thermal cycler must beat 94° C. when you put your samples inside.

[0365] Add Taq enzyme to the PCR mix just before you distribute 30 μl ofPCR mix per reaction.

[0366] PCR program: PTC-200 (MJ Research) or UnoII (Biometra)

[0367] step 1 3 min 94° C.

[0368] step 2 94° C. 30 sec.

[0369] step 3 55° C. 1 min 30 sec.

[0370] step 4 72° C. 3 min.

[0371] (31 cycles step 2 to 4)

[0372] step 5 72° C. 5 min.

[0373] step 6 15° C. for ever.

[0374] Check the quality, the quantity and the length of the PCRfragment on 1% agarose gel. Take 3 μl from the PCR reaction and mix with10 μl loading buffer (1.3 μl 10× blue loading buffer+8.7 μl H₂O; 10×blueloading buffer is: 10 mM Tris pHS, 1 mM EDTA, 30% glycerol, 0.2%bromophenol blue, 0.2% Xylene cyanol blue).

[0375] The length of the cloned fragment is the estimated length of thePCR fragment minus 300 bases that correspond to the amplified flankingplasmid sequences.

[0376] E: Sequencing PCR Fragments

[0377] Introduction

[0378] Two protocols are proposed, depending on the availability of anautomated sequencing machine. It should be noted that the quality of thesequence will depend greatly on the quality of the PCR experiment thatshould be carefully analyzed (number of bands and intensity). Smallamounts are generally sufficient as long as the preparation is not amixture of several fragments.

[0379] Manual Sequencing

[0380] Materials

[0381] Sequenase PCR Product Sequencing Kit (Amershamn/USB ref:US70170

[0382]³⁵S dATP (ICN Ref: 56420H; spec. Act: 1000 Ci/mmole)

[0383] 60 wells microtiter plate (Seromat Greiner ref:860173)

[0384] Primer MFR1 (10 picomoles/μl) 5′-GGC-TTA-CCC-ATA-CGA-TGT-TC-3′

[0385] 20×TTE buffer:

[0386] 216 g Tris Base

[0387] 72 g Taurine (USB/Amersham Ref: US75824)

[0388] 4 g NA₂EDTA,2H₂O

[0389] H₂O to 1000 ml

[0390] 40% Acrylamide (Eurobio Ref: 018806)

[0391] Urea (Prolabo Ref:28 877.292)

[0392] 10% Amonium Persulfate (APS)

[0393] TEMED

[0394] Experiment

[0395] (According to Amersham)

[0396] Enzymatic Pre-treatment of PCR Product

[0397] It is convenient to do this pre-treatment in a thermal cycler.

[0398] Take 7 μl of PCR amplification product. (Do not vortex the tubeto avoid taking cellular extract settling at the bottom of the tube).

[0399] Add 1 μl of ExoI (10 units) and 1 μl of Shrimp AlkalinePhosphatase (2 units). For large series, you can mix the 2 enzymes(vol/vol) and add directly 2 μl of the mix.

[0400] Gently mix the tube, briefly spin the tube and incubate at 37° C.for 15 min.

[0401] Inactivate the enzymes by heating at 80° C. for 15 min.

[0402] Chill on ice.

[0403] Denaturation and Annealing

[0404] Add 1 μl of primer to each tube and denature by heating 3 min. at100_(i)C.

[0405] Chill as quickly as possible in an ice/water bath for 5 min.

[0406] Centrifuge briefly and chill on ice.

[0407] Elongation

[0408] Mark the microtiter plate wells with the name of the clones.

[0409] For each template aliquot into microtiter plate wells 2 μl ofeach termination mix in the order GATC. Keep at 4° C. or on ice andpre-warm at 37° C. before use.

[0410] Prepare elongation mix, keep on ice; for every reaction you need:2 μl 5× sequenase buffer

[0411] 1 μl 100 mM DTT

[0412] 2 μl labeling mix (1:10 dilution)

[0413] 0.5 μl ³⁵S dATP

[0414] 2 μl Sequenase (add Sequenase just before use)

[0415] For example, for 6 reactions, prepare:

[0416] 14 μl 5×sequenase buffer

[0417] 7 μl 100 mM DTT

[0418] 14 μl labeling mix (1:10 dilution)

[0419] 3.5 μl ³⁵S dATP (5 μCi)

[0420] 14 μl Sequenase (just before use)

[0421] Set timer at 3 min.

[0422] Add 7.5 μl elongation mix to annealed template. Wait 30 sec.before continuing with next sample. You can do a maximum of 12 templatesin one go—yes, you have to work fast—keeping 15 sec. in between.

[0423] Aliquot 3.5 μl of elongation mix over each GATC termination wellswithin 30 sec (or 15 sec. when you do 12 templates in one go)

[0424] Wrap micro-titer plate with tape or para-film and put at 37° C.for 10 min. (can be placed in 37° C. incubator)

[0425] Stop termination by placing micro-titer plate on ice and adding 4μl stop-solution to each well

[0426] Denature samples just before loading:

[0427] Place micro-titer plate between two hot blocks at 95° C.; covertop of micro-titer plate with tissue to trap radio-active vapor. After 5min. start loading gel.

[0428] 6% Acrylamide Gel Electrophoresis

[0429] Apparatus: Sequi-GenII (Biorad 50 cm×40 cm).

[0430] The samples contain too much glycerol to use TBE buffer. Somedistortions could appear. The gels must run in 0.8× TTE buffer.

[0431] Dissolve 72.2 g urea with 59 ml d'H₂O, 6 ml of 20×TTE, 18.75 mlof 40% Acrylamide. Add 500 μl of 10% APS and 120 μl of TEMED.

[0432] Pre-run the gel for 30 min. at 150 Watts

[0433] Run the gel at 100 Watts

[0434] Abi Cycle Seqencing

[0435] Materials

[0436] 0.2 ml 8 tube-strips.

[0437] Exonuclease I (Amersham ref:E70073Z)

[0438] Shrimp Alkaline Phosphatase (Amersham ref: E70092Z)

[0439] Dye terminator (Perkin Elmer Ref: 402 122 or Amersham Ref:US79765)

[0440] Primer JC90 at 0.8 pmole/μl (see FIG. 8 for the map of pACTIIstplasmid)

[0441] 50 mg/ml of Dextran-Blue dissolved in 25 mM EDTA (pHS).

[0442] loading buffer: deionized formarnmide and Dextran-Blue in a ratioof 5:1 (formamide to Dextran-Blue).

[0443] 70% Ethanol in 0.5 mM MgCl₂.

[0444] 70% Ethanol in H₂O.

[0445] 40% Acrylamide (Biorad Ref: 161-0190)

[0446] Urea (Interchim Amresco Ref: 0568)

[0447] Experiment

[0448] Enzymatic Pre-treatment of PCR Product

[0449] It is convenient to do this pre-treatment in a thermal cycler(block of 96 wells) and to use 0.2 ml 8 tubes strips.

[0450] Take 6 μl of PCR amplification product. (Do not vortex the tubeto avoid taking the cellular extract at the bottom of the tube).

[0451] Add 1 μl of ExoI (10 units) and 1 μl of Shrimp AlkalinePhosphatase (1 unit). For large series, you can mix the 2 enzymes(vol/vol) and add directly 2 μl of the mix.

[0452] Briefly spin the tube (if necessary) and incubate at 37° C. for15 min.

[0453] Inactivate the enzymes by heating at 80° C. for 15 min.

[0454] Chill on ice.

[0455] PCR Sequencing

[0456] To each tube add 4 μl of primer at 0.8 pmole/μl and 8 μl ofTerminator Ready Reaction Mix.

[0457] Place the 8 tubes strips in the thermal cycler programmed for:

[0458] 10 sec. at 96° C.

[0459] 5 sec. at 50° C.

[0460] 4 min. at 60° C.

[0461] Repeat for 25 cycles.

[0462] 4° C. for ever.

[0463] Precipitate at room temperature (RT) each tube with 75 μl of 70%Ethanol in 0.5 mM MgCl₂.

[0464] Centrifuge at 3000 g for 15 min. at RT in a Jouan centrifuge(4000 rpm).

[0465] Discard carefully the supernatant with a Pasteur pipette undervacuum.

[0466] Wash with 200 μl of 70% Ethanol.

[0467] Discard carefully the supernatant with a Pasteur pipette undervacuum.

[0468] Dry the sample on the bench.

[0469] Resuspend the pellet with 5 μl of loading buffer by vortexing.

[0470] Spin the samples.

[0471] Heat the samples at 95° C. for 3 min. in the thermal cycler.

[0472] Load 2 μl of the samples on an acrylamide gel.

[0473] 6% Acrylamide Gel Electrophoresis

[0474] Dissolve 25 g urea with 19 ml of H₂O, 5 ml of 10×TBE, 7.5 ml of40% Acrylamide. Add 250 μl of 10% APS and 23 μl of TEMED.

[0475] Pour the gel according to Perkin Elmer.

[0476] Run gel on ABI 373 machine according to Perkin Elmer.

[0477] F: Identification and Classification of the Candidates

[0478] 1) Search in Databases

[0479] Introduction

[0480] This protocol varies from one lab to another depending on thelocal conditions for databases searches.

[0481] Materials

[0482] DNA Strider software (Marck. C; 16, 1829-1836, 1988)

[0483] SGD blast program (http://genome-www.stanford.edu; SGDB,Stanford, Calif.).

[0484] YPD (http://www.proteome.com; YPD, Proteome Inc., Beverley,Mass.).

[0485] NCBI blast program (http://www2.ncbi.nlm.nih.gov/BLAST).

[0486] Experiment

[0487] Sequence N-terminal Fusion of the Preys

[0488] Each clone is sequenced. Create one DNA Strider file per clone.

[0489] Read and enter the sequence from GGG ATC C. It is the BamHI siteused to clone the yeast FRYL library. Starting with the 3Gs gives thecoding frame of the Gal4 domain.

[0490] The 20 bases after the BamHI site are from the linker added tothe genomic DNA fragments for the making of the FRYL library.

[0491] It is important to check if the linker sequence contains amutation (especially frameshifts).

[0492] Read at least 50 bases of the insert.

[0493] Search chromosomal coordinates (chromosome number, strand w or c,position) with the SGD blast program.

[0494] Enter the data into in a column of an Excel Microsoft table.

[0495] Search the ORF corresponding to chromosomal coordinates (or thenames of the two flanking ORFs). Extract and copy the sequence of theORF into a DNA Strider file (+1000 nt flanking regions).

[0496] Biological information on the ORF can be extracted from the YPDdatabase.

[0497] Compare the insert sequence with the ORF sequence to get theexact location of the beginning of the insert related to the initiationcodon.

[0498] Enter the data in a column of the table.

[0499] Compare the translated sequence of the clones with the proteinsequence to check the coding frame.

[0500] Insert in a column of the table the length of the cloned fragmentas estimated by agarose gel electrophoresis of the PCR fragments.

[0501] 2) Classification of the Candidates

[0502] Considering the size of the library—a fusion point once everyfour bases—one can expect to find a given ORF several times asindependent clones in a complete screen of such a library. However, theprobability of the selection of a given fusion depends on the length ofthe interacting domain and on the position of the interacting domainalong the coding sequence.

[0503] These parameters predict that all candidates fall into one of thefollowing categories. The categories A1, A2, A3 and A4 correspond topotentially encoded yeast ORFs. Their inserts start either inside theORF coding sequence or upstream the initiation codon*.

[0504] The B category corresponds to fused polypeptides located in anintergenic region, in the reverse orientation of an ORF, in anon-polypeptide encoding region (rDNA, telomeric DNA, mitochondrial DNA)or in a Ty retrotransposon element.

[0505] The A1 category consists of candidates found several times asdistinct independent clones.

[0506] The three other A categories correspond to candidates found onlyas a single fusion, even if the same clone is found several times:

[0507] The A2 category consists of fusions starting close to aninitiation codon of a yeast ORF and at a distance smaller than 150 basesfrom the in-frame stop codon located upstream of this ORF. Thesecandidates correspond to amino-terminal interacting domains. For suchinteracting domains, fewer candidates are expected since in-framenonsense codons upstream of the yeast ORF interrupt its translation.

[0508] The A3 category candidates contain large coding inserts (over1000 bases). This category may correspond to preys with a largeinteracting domain. Since the average size of inserts is 700 nt,candidates with large interacting domains are underrepresented.

[0509] The A4 category contains the other candidates. We cannot predictwhy these clones are found only once, although several hypotheses can beproposed, such as incorrect folding or toxicity of fusion proteins.

[0510] G: Plasmid Rescue from Yeast by Eeletroporation

[0511] Introduction

[0512] This experiment allows the recovery of a plasmid from yeast cellsby transformation of E. coli with a yeast cellular extract. In thetwo-hybrid screening experiment, the diploid cells contain two differentplasmids carrying the TRP1 (bait) and the LEU2 (prey) markers,respectively. We use a bacterial strain (MC1066) that carries the trpand leu auxotrophies that can be complemented by TRP1 and LEU2 yeastgenes. Usually we select for the prey plasmid carrying LEU2 gene.

[0513] Materials

[0514] Plasmid Rescue

[0515] glass beads 425-600 μm (Sigma ref:G-8772)

[0516] Phenol/chloroform (1/1) premixed with isoamyl alcohol (Amrescoref: 0883)

[0517] Extraction buffer:

[0518] 2% Triton X100

[0519] 1% SDS

[0520] 100 mM NaCl

[0521] 10 mM TrisHCl pH8.0

[0522] 1 mM EDTA pH8.0

[0523] Mix Ethanol/NH₄Ac: 6 volumes Ethanol with 1 volume 7.5 M NH₄Acetate

[0524] 70% Ethanol

[0525] yeast cells in patches on plates.

[0526] note: This protocol can be performed with frozen cells preparedfrom colonies or patches on plate, mixed in water and frozen directly at−20° C. ( in 50 μl H₂O).

[0527] Electroporation

[0528] SOC medium

[0529] M9 medium

[0530] Selective plates: M9-Leu+Ampicillin

[0531] 2 mm electroporation cuvettes (Eurogentec ref: CE0002-25).

[0532] Experiment

[0533] Plasmid Rescue

[0534] First, add 400 μl of glass beads in each 1.5 ml eppendorf tube;second, add 200 μl extraction buffer (use multipipettor in case you do alarge number of extracts); third, put the cells of each patch in thetube using blue tips ( the cells must coat 2 mm of the tip).

[0535] resuspend the cells in extraction buffer with the blue tip.Continue under the hood.

[0536] Add 200 μl Phenol/chloroform and vortex cells vigorously for 7min.

[0537] Spin tubes 10 min, 15000 rpm.

[0538] Transfer 140 μl supernatant to a sterile eppendorf tube and addto each 500 μl Ethanol/NH₄Ac. Vortex.

[0539] Spin tubes 15 min 15000 rpm at 4° C.

[0540] Wash pellets with 250 μl 70% Ethanol. Dry pellets (5′ in SpeedVac).

[0541] Resuspend pellets in 10 μl water. Store extracts at −20° C.

[0542] Electroporation

[0543] For a large number of electroporations:

[0544] Take a large ice-bucket and line up the electrocuvettes accordingto their number.

[0545] Alongside the cuvettes line up sterile eppendorf tubes numberedas the cuvettes but also carrying the clone-name. To every tube add 1 μlof yeast plasmid DNA-extract.

[0546] Mark the selective plates M9-Leu with the date, the name of theclone as well as the number of the cuvette.

[0547] Fill sterile eppendorf tubes marked with cuvette number and clonename with 1 ml of SOC medium.

[0548] Thaw a vial with electrocompetent E.coli (Strain MC1066 forselection on Leu and Trp). Keep vial on ice.

[0549] To every 1 μl yeast DNA sample add 20 μl electrocompetent cells;mix and transfer the mix to the cold electroporation cuvette.

[0550] Do the electroporation directly. Set the Biorad electroporator on200 ohms resistance; 25 μF capacity; 2.5 Kvolts. Wipe cuvettes dry witha tissue paper and tap them to remove any trapped air-bubbles. Check ifsuspension makes contact with both electrodes. Place cuvette in thecuvette holder. Do the electroporation; time constant should be similarfor every electroporation (around 4.7).

[0551] Directly add 1 ml SOC into the cuvette and transfer the cell-mixinto sterile Eppendorf tube.

[0552] Let cells recover for 30 min at 37° C., spin the cells down 1min, 4000 g and pour off supernatant. Keep about 100 μl medium and useit to resuspend the cells and spread them on selective plates (e. g.M9-Leu plates). Pipet the suspension over the glass beads and directlyshake the plate to prevent a clustering of cells on the place where theyhave been pipetted (especially when plates are a bit dry).

[0553] Incubate plates for 36 h at 37° C.

[0554] Note: Wash the electrocuvettes and the caps with tap-water (applyhigh pressure to force out any remaining cells); rinse them with doubledistilled water and then With ethanol and let them dry. Close thecuvettes with the caps and store them in the supplier case according totheir number.

[0555] Preparation of Electrocompetent Cells

[0556] Inoculate 1 liter LB with a 10 ml o.n. pre-culture of the E.colistrain of interest (MC1066 for Two Hybrid-plasmid rescue)

[0557] Grow to an OD₆₀₀ of around 0.6. (It should not be higher than0.8).

[0558] Chill culture on ice and in cold-room for 15 min. From now onkeep cells cool and handle with care.

[0559] Pellet cells 15 min 3000 g at 4° C.

[0560] Resuspend cells in 1 liter cold sterile water

[0561] Pellet cells 15 min 3000 g at 4° C.

[0562] Resuspend cells in 500 ml cold sterile water

[0563] Pellet cells 15 min 3000 g at 4° C.

[0564] Resuspend cells in 20 ml sterile 10% glycerol

[0565] Pellet cells 15 min 3000 g at 4° C.

[0566] Resuspend cells in 2 ml sterile 10% glycerol and complete up to afinal volume of 3 ml (about 3.10¹⁰ cells/ml).

[0567] Distribute in aliquots of 50 μl or multiples of that. (You'lltake 50 μl per normal electroporation and 20 μl when you have to rescuea large number of plasmids)

[0568] Freeze the aliquots in dry Ice/EtOH but take care to preventtraces of EtOH entering into the tubes) and store at −80° C.

[0569] H: Plasmid Minipreps from E.coli

[0570] Materials

[0571] STET buffer:

[0572] 8% Sucrose

[0573] 50 mM EDTA pH8

[0574] 10 mM TrisHCl pH8

[0575] 0.5% Triton X100

[0576] 20 mg/ml freshly made Lysozyme (dissolved in water)

[0577] EtOH/NH₄Ac: mix 6 volumes of ethanol with 1 volume of 7.5 M NH₄Acetate.

[0578] 70% Ethanol

[0579] TE pH8

[0580] STET/Lysozyme: for 10 minipreps: 3 ml STET+250 μl 20 mg/mlLysozyme

[0581] Experiment

[0582] Start with 2 ml bacterial cultures grown overnight.

[0583] Transfer 1.5 ml to non-sterile Eppendorf tubes, pellet cells bycentrifugation 20 sec, discard the supernatant by overturning the tubes.Keep about 100 μl medium and use it to resuspend the cells with vortex.BE CAREFUL! all the cells must be well resuspended.

[0584] Add 300 μl STET/Lysozyme buffer

[0585] Keep on ice for 5 min.

[0586] Incubate 2 min at 100° C. (use hot-block rack for transfer).

[0587] Put on ice.

[0588] Spin 15 min at 4° C.

[0589] Take out the pellet with a toothpick

[0590] Add 750 μl EtOH/NH₄Ac to supernatant, vortex

[0591] Spin 10 min, wash with 500 μl 70% EtOH, dry pellet (5 min inspeed-vac).

[0592] Dissolve pellet in 50 μl TE by incubation at 65_(i)C for 5 minand vortex.

[0593] I: Yeast Transformation

[0594] Adapted from: Gietz et al, Yeast 11, 355-360, 1995

[0595] Material

[0596] 2 ml eppendorf tubes

[0597] 50 ml tubes

[0598] Media

[0599] liquid medium for preculture: YPglu or selective medium when theyeast strain harbors a plasmid.

[0600] plates for plasmid selection.

[0601] Solutions

[0602] Yeast carrier DNA (Clontech ref: K1606-A)

[0603] 1M Li Acetate (filter sterilized) (Fluka ref: 62395)

[0604] 10× TE (100 mM Tris pH 7.5; 10 mM EDTA) autoclaved

[0605] Sterile distilled water

[0606] Prepare 0.1 M LiAc/TE solution by mixing:

[0607] 100 ml 1M LiAc

[0608] 100 ml 10× TE

[0609] 800 ml sterile distillated water

[0610] 40% PEG 4000 (Merck ref:807 490) filtrated on Nalgene (0.2μ).

[0611] 40 g of PEG 4000

[0612] 10 ml 1M LiAc

[0613] 10 ml 10× TE

[0614] add H₂O to a final volume of 100 ml.

[0615] Experiment

[0616] For one transformation you need 10 ml of culture (OD=0.6; withour spectrophotometer, 1 OD corresponds to 10⁷ cells per ml) which willbe finally resuspended in 50 μl 0.1M LiAc/TE.

[0617] all steps are performed at room temperature

[0618] Set up a preculture of the strain in 20 ml of appropriate mediumthe day before

[0619] Grow o.n at 30° C. until saturation

[0620] Inoculate 50 ml of medium (for 5 transformations) at 0.15 OD withthe o.n preculture

[0621] Grow until OD=0.6

[0622] Centrifuge the cells in 50 ml sterile tubes at 5000 g (4000rpm)for 3 min

[0623] Discard the supernatant by overturning the tube

[0624] Resuspend the pellet with 2 ml of sterile water

[0625] Transfer into an 2 ml eppendorf tube.

[0626] Centrifuge the cells at 4000 g (6500 rpm) for 1 min.

[0627] Repeat the washing twice with 2 ml of water and twice with 2 mlof LiAc/TE solution.

[0628] Centrifuge at 4000 g 1 min. after each washing and resuspend eachtime the cells by vortexing.

[0629] Resuspend finally the pellet with 200 μl of LiAc/TE solution.Adjust to 250 μl.

[0630] In the meantime, mark your tubes. Do not forget one tube withoutplasmid DNA.

[0631] Distribute in sterile 1.5 ml Eppendorf tube:

[0632] 1 μl plasmid DNA (from a mini-prep or at 0.1 mg/ml)

[0633] 5 μl Yeast carrier DNA

[0634] 50 μl of cells

[0635] 350 μl of 40% PEG

[0636] Mix by inversion

[0637] Incubate 30 min at 30° C.

[0638] Heat shock cells at 42° C. for 20 min.

[0639] Add 700 μl of water. Mix by inversion.

[0640] Spin cells for 1 min in a microfuge at 4000 g. Discardsupernatan.

[0641] Resuspend the cells with 100 μl of water

[0642] Spread the cells on appropriate medium plates and incubate at 30°C. for two-three days.

[0643] J: Characteristics of the FRYL Library

[0644] Origin of the plasmid: pACTIIst.

[0645] Origin of the genomic DNA: strain Ym955 (a gift of M. Johnston).

[0646] Ym955 genotype: Mat a ura3-52, his3-200, ade2-101, lys2-801,leu2-3,112, trp1-901, tyr1-501, gal4-542, gal80-538.

[0647] Note: his3-200, trp1-901, gal4-542 and gal80-538 are deletions ofthe whole coding sequence.

[0648] Library Construction into E.coli

[0649] FRYL Genomic library was made according to the procedure ofElledge et al (P.N.A.S., 1991, 88, 1731-1735).

[0650] Genomic DNA was sonicated, made blunt by 3 modification enzymes(Mung bean nuclease, T4 DNA Polymerase and Klenow fragment). Adaptorswere ligated to blunt ends.

[0651] Adaptors were designed to allow blunt ligation at one extremityand cohesive ligation with a 3 nucleotides overhang at the other end.

[0652] Sequence of adaptors:

[0653] 5′-ATCCCGGACGAAGGCC-3′

[0654] 5′-GGCCTTCGTCCGG-3′

[0655] Only the former was phosphorylated before annealing to avoidself-ligation of the adaptors. After ligation the inserts were purifiedfrom free adaptors and small fragments on a Chroma Spin column(Clontech).

[0656] The pACTIIst vector was digested with BamHI and the extremitieswere filled-in with dGTP by the Vent (Exo⁻) polymerase (New EnglandBiolabs), generating extremities complementary to the 3 nucleotidesoverhang of adaptors but preventing self-ligation of the vector. (BamHIsites are reconstituted at each end of the insert).

[0657] Inserts and vectors were ligated together and ligation productswere used to transform E.coli MR32.

[0658] 5.10⁶ clones were obtained. All transformants were scraped fromdishes and the pool of transformants was frozen in LB/glycerol. Thetiter of the library is 1-2. 10⁹ transformants/ml.

[0659]E. coli FRYL Library Characteristics

[0660] 5 10⁶ clones

[0661] 72.5% with insert i.e 3.6×10⁶ clones.

[0662] mean length of DNA fragments: 700 bp

[0663] 3.6×10⁶ clones represent 3.6×10⁶ independent genomic fragmentsi.e. 3.6×10⁶ random fusion points in the genome.

[0664] Yeast genome: 14×10⁶ bases

[0665] In the FRYL library, the yeast genome is cut every 4 bases(14×10⁶ divided by 3.6×10⁶)

[0666] FRYL Library Transformed in the Y187 Yeast Strain

[0667] The Y187 yeast strain is transformed according to standardprocedures with the FRYL library DNA. As many as possible transformedyeast colonies are collected and pooled. Aliquots are stored at −80° C.

[0668] Typically, several hundreds plates are used to allow the growthof 10.000 to 30.000 colonies per plate (90 mm diameter). Ideally, threetimes more yeast colonies than the initial number of E. coli clonesshould be collected. The same calculation occurs to estimate how manyyeast diploid cells should be screened to cover the original library.

[0669] FRYL E. coli library: 5.10⁶ clones.

[0670] FRYL yeast frozen library: around 15.10⁶ clones.

[0671] Diploids to be tested: 45.10⁶ cells.

[0672] The mating efficiency is usually above 20% and often around 50%.

[0673] 1 Vial should contain at least 4.10⁸ cells

[0674] Theoretical calculation:

[0675] (1−p)=(1−1/n)^(N)

[0676] N=number of clones that are tested (number of yeast colonies)

[0677] n=number of original clones (number of E. coli clones)

[0678] p=probability to test a given clone of the original library

[0679] For example: with n=5.10⁶ E. coli clones N p 23.10⁶ 99% 15.10⁶95% 13.10⁶ 93% 7,6.10⁶ 78% 7,15.10⁶ 76%

[0680] K: Gene Disruption and Complementation

[0681] Gene disruptions were performed with a TRP1 cassette replacingthe entire ORF (Baudin et al., 1993). BMA64 diploid cells weretransformed with a PCR-derived linear DNA fragment, then transformantswere re-isolated and disruptions were controlled by Southern analysis.After tetrad dissection, spores were incubated on rich medium at 23° C.for three days and clones were then replica-plated on rich medium andon—W plates and incubated at 23°, 30° and 37° C. For those genes foundessential, the diploid strain was transformed with a plasmid encoding aGal4 fusion protein selected during the two-hybrid screen. Tetrads werethen dissected in order to test the complementation of the disruption bythe expression of the fusion gene.

[0682] L: Construction of the FRYL Library

[0683] Protocols

[0684] Step 1. Preparation of yeast genomic DNA from Ym955 strainaccording to standard procedure;

[0685] Origin of the genomic DNA: Ym955 (a gift of M. Johnston).

[0686] Ym955=ura3-52, his3-200, ade2-101, lys2-801, leu2-3,112,trp1-901, tyr1-501, gal4-542, gal80-538.

[0687] his3-200, trp1-901, gal4-542 and gal80-538 are deletions of allcoding sequences.

[0688] Step 2. Preparation of randomly sheared yeast DNA by sonication

[0689] 44 μg of yeast genomic DNA in a volume of 100 μl were sonicatedto an average size of 1000 bp (range size 200 bp-2000 bp).

[0690] Step 3. Blunting the DNA fragments ends with Mung-Bean nucleasetreatment and T4 DNA polymerase repair.

[0691] Yeast sheared DNA at a concentration of 0.2 mg/ml was treatedwith 40 units of Mung-Bean nuclease in a final volume of 200 μl for 30′at 30° C.

[0692] Phenol-chloroform extraction and precipitation with ethanol.

[0693] Dissolved the sheared DNA (40 μg) in 143 μl of H₂O; Treated with40 units of T4 polymerase, 100 μM dNTP in a final volume of 200 μl for10′ at 37° C.

[0694] Added 11 units of Klenow enzyme and incubated for 10′ at roomtemperature and 1 hr at 16° C.

[0695] Phenol-chloroform extraction and DNA precipitation with ethanol.

[0696] Dissolved the precipitated DNA in 10 μl of H₂O.

[0697] Step 4. Preparation of the adapters for ligation with the shearedyeast DNA

[0698] Mixed

[0699] 3 μg oligo 160

[0700] 3 μl ATP 10 mM

[0701] 3 μl 10× T4 polynucleotide buffer

[0702] 1.5 μl T4 polynucleotide Kinase (10 u/μl)

[0703] H₂O qsp 30 μl

[0704] Incubated 30′ at 37° C. followed by 5′ at 95° C. Added 15 μl (30μg) of oligo 159

[0705] Incubated: 5′ at 95° C., 10′ at 68° C., 15′ at 42° C., 10′ atroom temperature.

[0706] Sequence of the oligo pL 160: 5′-ATCCCGGACGAAGGCC

[0707] Sequence of the oligo pL 159 5′-GGCCTTCGTCCGG

[0708] Step 5. Ligation of adaptors to the sheared DNA

[0709] Added to the 10 μl of sheared yeast DNA from step 3: 38 μl ofadaptors from step 4, 6 μl 10×ligation buffer, 3 μl BSA 1 mg/ml, 0.6 μlDTT 1M, 1.2 μl ATP 10 mM, 1 μl T4 DNA ligase (Biolabs 2000000 U/μl).

[0710] Incubated at 4° C. overnight.

[0711] Added 1 μl T4 DNA ligase and incubated 24 hrs at 15° C.

[0712] Added 40 μl of H₂O to the ligation

[0713] Step 6. Purification of the DNA from unligated adaptors and itssize fractionation on a Chroma-spin 400 column (Clontech ref:K1323-1)

[0714] Split the solution into two aliquots of 50 μl each.

[0715] Fractionated one aliquot on a chroma spin 400 column according tothe manufacturer instructions

[0716] Collected the 1st fraction from the column in a final volume of50 μl.

[0717] Preparation of the vector pACTII st

[0718] Step 7. Digestion vector with the restriction enzyme BamHI

[0719] Digested to completion 20 μg of pACTIIstop vector with BamHIrestriction enzyme in a final volume of 100 μl at 37° C.

[0720] The vector has been phenol-chloroform extracted and precipitatedwith ethanol.

[0721] Step 8. Dephosphorylation of the vector ends

[0722] 20 μg of pACTIIst from step 7 were dephosphorylated with 3 unitsof calf intestinal alkaline phosphatase in a final volume of 110 μl for1 hr at 37° C.

[0723] The phosphatase was inactivated by adding 1 μl of a 0.5 Msolution of EDTA ph 8 and incubation 10′ at 75° C.

[0724] The solution was phenol-chloroform extracted and the DNArecovered by ethanol precipitation. The DNA was resuspended with 100 μlof TE pH8

[0725] Step 9. G-filling-in of the BamHI cut-vector

[0726] To fill-in the ends of the vector with dGTP the followingreactions were set up:

[0727] 50 μl (10 μg) pACTIIst cut BamHI from step 8

[0728] 15 μl Vent polymerase buffer 10×

[0729] 6 μl dGTP 10 mM

[0730] 5 μl triton X100

[0731] H₂O qsp 145 μl

[0732] Incubated the solution 5′ at 72° C.

[0733] Added 5 units of exo⁻ Vent DNA polymerase

[0734] Incubated 1′ at 72° C.

[0735] Put the reaction on ice

[0736] The DNA was extracted with phenol-chloroform and recovered byethanol precipitation.

[0737] Dissolved pACTIIst in 100 μl of TE ph 8.

[0738] Step 10. Ligation sheared yeast genomic DNA from step 6 intopACTIIst vector from step 9

[0739] The two following reactions were set up to ligate an aliquot ofrandomly sheared yeast DNA to the pACTIIst vector:

[0740] 50 μl pACTIIst vector (5 μg) from step 9

[0741] 25 μl yeast genomic DNA (5 μg) from step 6

[0742] 50 μl T4 ligase buffer 10× (Biolabs)

[0743] 365 μl H₂O

[0744] 2 μl T4 ligase 2000000 u/μl Biolabs

[0745] Incubate the reaction overnight at 15° C.

[0746] the ligation reaction was precipitated with ethanol

[0747] Step 11. Preparation of the library into E. coli strain MR32

[0748] The ligation was resuspended in 50 μl of H₂O. Each 2,5 μl ofligation were independently electroporated into 50 μl ofelectrocompetent E.coli strain MR32 cells. Each electroporated tube wasdiluted in 1 ml SOC buffer. The 20 ml of transformants were pooled. 50μl were plated on LB plate+ampicillin to get a density of 13000-20000cfu/plate.

[0749] The library into the pACTIIst vector consists of 5.10⁶independent transformants.

[0750] Transformants of the library into pACTIIst were washed away with3 ml/plate. 750 ml of LB medium at a concentration of 43 OD₆₀₀/ml werecollected.

[0751] Aliquots of the bacterial FRYL library were stored at −80° C.(2.10⁹ cells/ml/vial).

[0752] The rest of the cells was collected by centrifugation and thecell pellet used for plasmid preparation.

[0753] Step 12. Plasmid library preparation

[0754] Plasmids were collected using Qiagen columns according tomanufacturer instructions.

[0755] The plasmid library was dissolved at a concentration of 1 mg/ml.

Preparation Library into Yeast Strain Y187

[0756] Step 13. Yeast transformation

[0757] 400 μg of plasmid library were used to transform 1 liter of Y187yeast strain cells at a concentration of 0.9 OD₆₀₀/ml according tostandard conditions.

[0758] Transformants were plated on solid synthetic media lackingleucine at a density of 10000 cfu/plate

[0759] 13×10⁵ independent transformants were obtained for the FRYL1library.

[0760] Y187 transformants that constitute the library were collected in1400 ml of YPGlu at a concentration of 90 u. OD600/ml; 40 g. of 100%glycerol were added per 100 ml of yeast transformant solution and thelibrary was aliquoted in 1212 vials at 1,5 ml/ vial (4×10⁸ cells/vial)and stored at −80° C.

[0761] M: MEDIA

[0762] Yeast Media

[0763] YPGLU

[0764] 1% yeast extract

[0765] 2% bactopeptone

[0766] 2% glucose

[0767] 10 g Bactopeptone (Difco ref: 0118-17-0)

[0768] 10 g Yeast Extract (Difco ref: 0127-17-9)

[0769] 20 g Glucose (Merck ref: 1.08342)

[0770] Fill to 1 liter with distillated water

[0771] Shake until all ingredients are dissolved

[0772] For plates add 20 g Bacto-agar (Difco ref:0140-01)

[0773] Autoclave 20 min, 110° C. (at higher temperatures sugars andamino acids might be degraded), let cool to 60° C., (when required addantibiotics) mix gracefully so that no bubbles are formed Pour sterileplates: 25 ml per plate

[0774] Drop-Out Yeast Medium

[0775] 6.7 g Yeast Nitrogene Base w/o amino acids (Difco ref:0919-15-3)

[0776] 20 g D-glucose (Merck ref:1.08342)

[0777] 2 g drop-out powder mix (see below)

[0778] Fill to 1 liter with distilled water

[0779] Shake/stir until all ingredients are dissolved

[0780] For plates: add 20 g bacto-agar (Difco ref:0140-01), mix

[0781] Autoclave 20 min, 110° C. (at higher temperatures sugars andamino acids might be degraded), let cool to 60° C., (when required addantibiotics) mix gracefully so that no bubbles are formed

[0782] Pour sterile plates: 25 ml per plate

[0783] The four Two-hybrid drop-out mixes are:

[0784] (selection on bait plasmid);

[0785] L (selection on library plasmid);

[0786] LW (selection on both plasmids);

[0787] LWH (selection for both plasmids and two-hybrid interactioninducing the HIS3 reporter).

[0788] LWH+AT (AT may be added at a concentration between 1 mM to 50 mMdepending on the bait)

[0789] Tetracycline (12 mg/ml stock solution) is added after cooling theagar to 60° C.

[0790] 3-Amino 1,2,4 Triazole (3-AT Sigma Ref: A-8056) is added aftercooling the agar to 60° C. using a 1M stock solution.

[0791] Drop-out Powder Mix

[0792] Mix 2 g of each component:

[0793] Adenine; Alanine; Arginine; Asparagine; Aspartic acid; Cystein;Glutamic acid; Glutamine Glycine; Histidine; Isoleucine; Lysine;Methionine; Phenylalanine; Proline; Serine; Threonine; Tyrosine;Tryptophan; Uracil; Valine

[0794] plus 4 g of Leucine.

[0795] In total 22 essential components from which the underlined oneshave to be omitted in the case of the two-hybrid drop-out mixes.BACTERIAL MEDIA SOC BUFFER 2% Tryptone (20 g/l) 0.5% Yeast Extract (5g/l) 10 mM NaCl (0.58 g/l) 2.5 mM KCl (0.19 g/l) 10 mM MgCl₂ (2.03 g/l)10 mM MgSO₄ (2.46 g/l for MgSO₄.7H₂O) 20 mM glucose (3.6 g/l)

[0796] M9 Drop-out Plates

[0797] 1 g drop-out mix (the same as for yeast medium) (for libraryplasmids M9-leu: -L; for bait plasmids M9-Trp: -W)

[0798] 20 g bacto agar in 878 ml double distilled water,

[0799] Autoclave for 20 min at 110° C.

[0800] Let cool to 60° C.;

[0801] Add asceptically:

[0802] 100 ml 10× M9 (autoclaved)

[0803] 10 ml 20% glucose (autoclaved)

[0804] 2 ml 1M MgSO₄ (autoclaved)

[0805] 10 ml 20 mM CaCl₂ (autoclaved)

[0806] 1 ml 1000× Ampicilline (100 mg/ml stock)

[0807] Pour plates (20 ml/plate)

[0808] for 1 liter of 10× M9:

[0809] 60 g Na₂HPO₄;

[0810] 30 g KH₂PO₄;

[0811] 5 g NaCl;

[0812] 10 g NH₄Cl

[0813] Adjust to pH 7.4

[0814] One plate is needed for every rescue

EXAMPLES Example 1 Complete Yeast Genome Screening in a Two-hybridStrategy

[0815] We constructed a yeast genomic DNA library into a derivative ofthe pACTII two-hybrid bait vector (Clontech). The genomic DNA wassonicated and cloned following a procedure that prevents the cloning ofmultiple fragments in the same plasmid (see Experimental Procedures,Elledge et al., 1991). We recovered 5.10⁶ independent E. coli clonesthat were pooled to constitute the FRYL library. We analyzed 40independent clones: 72% of them contained an insert 200 to 1400 nt long.The average size of the inserts was 700 bp. Considering the size of theyeast genome (14.10⁶ bp), a fusion event occurs statistically once everyfour bases, leading to an in frame fusion between the Gal4 activationdomain and a yeast ORF once every 24 nucleotides. One can expect to findseveral times a given ORF as independent clones in a complete screen ofsuch a library. However, the probability of the selection of a givenfusion depends on the length of the interacting domain and on theposition of the interacting domain along the coding sequence.

[0816] These parameters predict that all candidates fall in 5 categories(FIG. 1). The four A categories correspond to yeast ORFs. The B categorycorresponds to fused polypeptides located in an intergenic region, inthe reverse orientation of an ORF, in a non-polypeptide encoding region(rDNA, telomeric DNA, mitochondrial DNA) or in a Ty retrotransposonelement. A1-A4 candidates encode potentially interesting fusedpolypeptides. Their inserts start either inside the ORF coding sequenceor upstream the initiation codon. The A1 category consists of candidatesfound several times as independent clones. The three other A categoriescorrespond to candidates found only as a single fusion, even if the sameclone is found several times. The A2 category consists of fusionsstarting close to an initiation codon of a yeast ORF and at a distancesmaller than 150 bases from the in-frame stop codon located upstreamthis ORF. These candidates correspond to amino-terminal interactingdomains. For such interacting domains, fewer candidates are expectedsince in-frame non sense codons upstream of the yeast ORF interrupt itstranslation. The A3 category candidates contain large coding inserts(over 1000 bases). This category may correspond to preys with a largeinteracting domain. Since the average size of inserts is 700 nt,candidates with large interacting domains are underrepresented. The A4category contains the other candidates. We cannot predict why theseclones are found only once, although several hypotheses can be proposed,such as incorrect folding or toxicity of fusion proteins (Fields andJang, 1996). Most candidates in the A categories are fused to the Gal4sequence in the reading frame of the yeast gene. However, several ofthem are out of frame and we show that, at least in some cases,frameshifted translation occurs (see below). In the Tables, the out offrame candidates are labeled with an asterisk.

[0817] The classification of interacting candidates found in atwo-hybrid screen requires a reproducible experimental protocol thatallows a complete coverage of the library. For this purpose, wedeveloped a mating strategy (FIG. 2; see also Experimental Procedures).In brief, we transformed the Y187 strain with the FRYL library DNA.Thirteen million yeast colonies, corresponding to an almost completecoverage of the E. coli library were recovered, aliquoted and frozen.Independently, bait plasmids were introduced in the CG1945 strain. TheY187 strain contains a sensitive LacZ reporter gene whereas the CG1945strain has a non-leaky HIS3 reporter gene that enables the selection ofpositive diploids in the absence of 3-aminotriazol (3-AT). The matingprocedure allows a direct selection on selective plates because the twofusion proteins are already produced in the parental cells. No replicaplating is required. We routinely obtain a 20% mating efficiency andregularly up to 50% mating efficiency. Screening forty million diploidsensures the coverage of the yeast library. The X-Gal assay is directlyperformed on His⁺ colonies. Prey plasmids from all colonies aresequenced at the Gal4 junction.

[0818] The bait plasmid is one of the key parameters for a two-hybridscreen. The pAS2 vector encodes a GAL4-DNA binding domain. It was foundvery efficient for protein-protein interaction analyses (Legrain et al.,1994). However, the CYH2 gene was found toxic when overexpressed(roughly 10% plating efficiency for cells containing this plasmid, asopposed to 80% for cells transformed with a derivative of pAS2 deletedfor the CYH2 gene, data not shown). This is a serious limitation forlarge scale screens. In addition, poor specificity was observed inscreens due to the presence of the HA epitope. After deletion of thissequence, we obtained 10 times less His⁺, LacZ⁺ colonies in a two-hybridscreen with Prp21p (data not shown). For these reasons, baits werecloned as full length ORFs into the pAS2ΔΔ vector which was devoid ofthe CYH2 gene and the HA epitope (see Experimental procedures).Expression of the fusion proteins was controlled by western blot (FIG.3). Fusion proteins were detected roughly at the expected molecularweight.

Example 2 Exhaustive Two-hybrid Screens with Yeast Proteins Are HighlySelective and Point Toward Limited Sets of Interacting Proteins

[0819] The ten proteins chosen as initial baits in two-hybrid screensare nuclear proteins implicated in the pre-mRNA splicing pathway (Table1). Smd1p and Smd3p are core proteins of the snRNP particles, Snp1p andMud1p are specific proteins of the U1 snRNP (yeast homologues of humanU170K and U1A, respectively), Mud2p is associated with the U1 snRNPduring early steps of spliceosome assembly, Yir009w is a U2 snRNPparticle protein (the yeast homologue of the human U2B″), Prp9p, Prp11pand Prp21p are the three components of the yeast SF3a factor associatedwith the U2 snRNP particle and Cus1p is the first identified componentof the yeast SF3b factor, also associated with the U2 snRNP (see forreview Beggs, 1995; and also Tang et al., 1996; Wells et al., 1996).

[0820] The number of His⁺ colonies varied considerably among the variousscreens (58 to 17000, Table 1). However, the number of His⁺, LacZ⁺colonies was less variable (10-86), allowing a selection of roughly onepositive clone for one million diploid (ranging from 0.1 to 4 permillion). The number of recovered and identified candidates was usuallyvery close to the initial number of His⁺, LacZ⁺ colonies (Table 1).Thus, the collection of identified clones is a fair representation ofthe initial clones selected in the screens. Preys were sequenced andgrouped according to the classification described above (FIG. 1). Asillustrated in FIG. 4, the various baits behave differently. The ratioof identified loci out of the number of selected preys varied from baitto bait (e.g. 8 out of 61 in the Mud2p screen as compared to 30 out of46 in the Snp1p screen). In addition, the categories of preys weredifferently represented in screens selecting few clones (e.g. 13 preysfalling in two A1, two A2 and two A4 in the Cus1p screen and 9 preysfalling in two A2, three A4 and one B in the Mud1p screen). Finally, wenote that B loci can be efficiently selected as multiple independentfusions. The most likely interpretation is the selection of a givenartificial polypeptide that interacts strongly with the bait. Within ascreen, typically two or three ORFs fall into the A1 category. Manypreys are proteins of unknown function and some of them were in turnused as baits (Table 1). They also behaved differently from one another(FIG. 4), but in every case, few ORFs were selected again, allowing thechoice of a novel prey as bait for a third round of screening.

Example 3 Two-hybrid Screens with Known Proteins Identify NovelProtein-protein Interactions with Potential Biological Significance

[0821] The first round of screening was done with known splicingfactors. All selected ORFs are listed in the following tables accordingto the official yeast ORF nomenclature with their gene name and theirfunction, when available (Tables 2 to 7, Garrels, 1996). The category ofcandidates (FIG. 1) is given for each ORF.

[0822] The Smd1p and Smd3p screens select 8 and 11 different ORFsrespectively, with two A1 candidates in each case (Table 2). One A1Smd3p candidate is Yer029c, a protein that exhibits Sm motifs shared bycore proteins of snRNP particles (Hermann et al., 1995; Seraphin, 1995).This new protein was used for a second round two-hybrid screen (seebelow).

[0823] Screens performed with the two U1 snRNP-associated proteins,Snp1p and Mud1p, reveal two extreme situations (Table 3). We found 42clones in 28 different genetic loci with Snp1p as bait, including fiveA1 ORFs. Among the A1 ORFS there were transcriptional factors, includingSwi1p, that were also obtained with other baits (see below). Incontrast, only eight candidates falling in five genetic loci were foundwith Mud1p, none of them falling in the A1 category. None was consideredfor further screening.

[0824] The Mud2p screen is very striking (Table 4): only seven ORFs wereselected, one of them, Ylr116w, represented by more than 60% of theclones. This unknown yeast gene was chosen as bait in a second roundtwo-hybrid screen. Two other candidates of unknown functions are theLpg4p and the Smy2p proteins, which are highly homologous with eachother.

[0825] The Yir009w (U2BÕÕ) screen identified two genetic loci in the A1category (Table 5). One of them, YLR456W, encodes a protein of unknownfunction that was chosen as a second round bait.

[0826] Screens were also performed with Prp9p, Prp11p and Prp21 p (Table6). Prp9p and Prp 11p were previously shown to interact with Prp21 paltogether forming the yeast homologue of the human SF3a splicing factor(Bennett and Reed, 1993; Brosi et al., 1993; Legrain and Chapon, 1993).The Gal4-Prp9p fusion could not be used due to a very strong directactivation of reporter genes, and the two-hybrid screen was performedwith the LexA-Prp9p fusion as bait and in the presence of 5 mM 3-AT toeliminate a high background of residual growth. Still the expected PRP21prey was found among 14 different genetic loci. The Prp11p screenselected 17 different ORFs. Five of them correspond to the A1 category,Prp21p being the most represented. The Prp21p screen confirms knowninteractions with the two other SF3a subunits: Prp9p and Prp11p arefound among the 13 different ORFs. Prp11p is the only A1 candidate. Notethat Prp9p which interacts with Prp21p in its amino-terminal domain(Legrain et al., 1993) was selected as an A2 candidate, illustrating ourprediction regarding this category. Finally, we note the presence of acommon A2/A3 candidate, Ynr053c, in the Prp9p, Prp11p and Prp21pscreens. The YNR053C gene contains an intron downstream of a large exon1 (810 nt) and we always select the same fusion including the two exons.

[0827] The Cus1p screen selected thirteen clones, all falling in sixyeast ORFs (Table 7). Yor319w found as an A2 candidate exhibits a stronghomology with SAP49, one of the human SF3b components (Wells et al.,1996) and was chosen as a second round bait.

Example 4 Second and Third Round Two-hybrid Screens with UnknownProteins Reveal Novel Interactions with Relevant Known Proteins

[0828] Four proteins of unknown functions selected as A1 or A2candidates were chosen for second round screening (Table 8). In threescreens (Yer029c, Ylr456w and Ylr116w) the initial bait (SmD3p, Yir009wand Mud2p, respectively) was not found in the set of preys. In addition,in Yer029c and Ylr456w screens, no A1 candidate was found and no furtherexperiments were performed with these proteins. The Ylr116w screenselected 11 ORFs, including two A1 (Smy2p and Lpg4p). Two splicingfactors, Prp39p and Prp22p, were found among the preys. Thus, an unknownprotein, Ylr116W, selected as prey with a splicing factor, Mud2p, can inturn select splicing factors, strongly suggesting that Ylr116w itselfparticipates in the splicing pathway. Furthermore, the Mud2p and Ylr116wscreens have several preys in common, including Smy2p, Lpg4p and Prp39p,suggesting that these five proteins participate in a common complex.

[0829] The Yor319w screen selected 9 preys falling in 4 different ORFs,two being in the A1 category. One of them is Cus1p, confirming theinitial interaction detected with this protein as bait (Table 7). Thesecond A1 candidate is Yjr022w. A careful analysis of its aminoacidsequence revealed that this protein contains the Sm motifs common to thecore proteins of the snRNP particles (see Discussion). We used thisYjr022w protein as a bait for a third round two-hybrid screen (Table 9).Nineteen ORFs were selected, including the initial bait that selectedYjr022w (Yor319w). We also find two other Sm proteins including Uss1p, aU6 snRNP-associated protein (Cooper et al., 1995). This result suggestsa connection between the U2 and U6 snRNPs (see Discussion).

[0830] A two-hybrid assay was also systematically performed withLexA-fusion baits for all preys selected in the Gal4-fusion screens(Tables 2-9). The LexA-fusion assay allows us to check whether thetwo-hybrid positive signal is independent from the DNA binding domainused as a fused polypeptide. Most preys selected by a Gal4-fusion werepositive in the LexA-fusion assay, except for the Yir009w and theYjr022w screens (6 and 9 negative preys out of 19, respectively). Thus,preys are mostly selected by the intrinsic domain of the bait fused tothe Gal4 domain and not by the Gal4 domain itself or by a possible hingedomain formed in the fusion protein. However, we note that theexperimental conditions for this two-hybrid assay are probably lessstringent than those used during the screening procedure (seeExperimental Procedures).

[0831] Altogether, 170 preys were selected in the 15 screens andcorrespond to 145 different ORFs, implicated in different cellularpathways. Nine ORFs are involved in pre-mRNA splicing, six in other RNAmetabolisms, ten are involved in transcription and four are proteinkinases or phosphatases. Other known ORFs are proteins implicated inmany different functions. Fifty per cent of the selected ORFs correspondto new proteins for which one now has a starting point for a functionalanalysis. This proportion is similar, whether the screen is performedwith a known protein (first round) or not (second and third round).Finally, among the selected ORFs, we note the presence of five unknownORFs which exhibit homology with known human splicing factors. Ylr116wand Yor319w were recently found homologous to human splicing factors SF1and SAP49, respectively (Arning et al., 1996; Wells et al., 1996) andYer029c, Yjr022w and Yb1026w/Snp3p exhibit Sm motifs found in snRNP coreproteins (see Discussion).

Example 5 Overlapping Fragments Selected in Two-hybrid Screens DefineInteracting Domains

[0832] A1 candidates correspond to independent fusions coveringfragments of the same ORF. The comparison of the 5Õ and 3Õ junctions ofthese fusions allows the definition of a minimal fragment necessary forthe interaction with the bait protein. Such an example for Smy2p andLpg4p, two preys selected in the Mud2p and the Ylr116w screens, isillustrated in FIG. 5A. All clones share a common region. The deducedminimal fragment in Smy2p required for the interaction with Ylr116wcovers 254 aminoacid residues (aa 191-402). Only one SMY2 fusion wasselected in the Mud2p screen (aa 14-402). Similarly, the minimalfragments of Lpg4p sufficient for the interaction with Mud2p and Ylr116wcan be determined: 113 residues (aa 150-262) and 129 residues (aa140-268), respectively. In addition, Smy2p preys selected in the Ylr116wscreen also interact with Mud2p and the smallest Lpg4p prey selected inthe Mud1p screen interacts with Ylr116w (data not shown). This leads toa minimal size of 212 residues (aa 191-402) and 113 residues (aa150-262) for the interacting domains in the Smy2p and Lpg4p proteins,respectively. Taking into account the fact that Smy2p and Lpg4p arehomologous over their entire sequence (similarity: 55%; identity: 38%),our results strongly suggest that Lpg4p and Smy2p share a common domainthat interacts with Mud2p and Ylr116w.

[0833] Mud2p and Ylr116w also interact together (Table 4). Ylr116w andits human homologue SF1 contain a KH domain (FIG. 5B, Arning et al.,1996). The minimal interacting domain of Ylr116w with Mud2p (aa 41-141)is located upstream the KH domain and corresponds to a very conservedregion between the yeast and human proteins (FIG. 5B).

[0834] Prp9p and Prp11p were previously shown to interact with Prp2 1pthrough domains that were defined by point mutant and deletion mutantanalyses (Legrain and Chapon, 1993; Legrain et al., 1993; Rain et al.,1996). Here we determine more precisely the interacting domains ofPrp21p with Prp9p and Prp11p and of Prp11p with Prp21p (data not shown).Similarly, we also characterize an interaction between Cus1p and a novelsubunit of the yeast SF3b, Yor319w. The interacting domain is located inthe carboxy-terminal region of Cus1p (aa 244-436). In conclusion, thecharacterization of A1 inserts selected in two-hybrid screens allows thedelineation of a minimal fragment necessary for interaction.

Example 6 Many Novel ORFs Revealed in Two-hybrid Screens Are Essentialfor Viability

[0835] Many selected preys correspond to new uncharacterized ORFs. Wedisrupted fifteen of these ORFs selected as A1, A2 or A3 candidates.Seven disrupted strains exhibit no growth phenotype (YDR386W, YJR033C,YJR039W, YLR456W, YMR285C, YNL023C, YPL105C), one has a slow growthphenotype (YLR357W) and seven disruptions are lethal (YDR180W, YER029C,YHR197W, YJR022W, YLR116W, YMR117C, YNR053C). This list of new essentialproteins includes i) Ynr053c, that was selected in several two-hybridscreens (Tables 6, 8 and 9), ii) one new splicing factor: Ylr116w, theyeast homologue of human SF1, and iii) Yer029c and Yjr022w which exhibitSm motifs. The three latter disruptions can be complemented with fusiongenes selected in the two-hybrid screens, indicating that the Gal4 fuseddomain has no detrimental effect and that these fusion proteins can beused as tagged proteins.

Example 7 Out of Frame Fusion Genes Are Selected in Two-hybrid Screensand Produce Frameshifted Polypeptides

[0836] Among the 29 A1 ORFs selected in the 15 screens, nine includeinserts with out of frame fusions, two of which only represented as outof frame fusions (see Tables 2-9). This result demanded a more carefulanalysis. Eight inserts overlapping the YPL105C ORF were selected in theYlr116w screen (FIG. 5A). Five out of eight inserts are fused out offrame with the Gal4 domain (three in the −1 frame and two in the +1frame). In both groups, different polypeptides are produced whentranslation occurs in frame with the Gal4 domain. Similarly, all insertsoverlapping with the YEL015W ORF selected in the Yjr022w screen are inthe −1 or in the +1 frame and encode different polypeptides in the Gal4frame (data not shown). Statistically, the selection of these inserts atthe same locus via the production of different polypeptides is mostunlikely. It certainly occurs through the production of frameshiftedpolypeptides.

[0837] This conclusion is supported by two independent experiments offunctional complementation. Genetic defects were corrected by theexpression of a frameshifted fusion ORF. Prp11p was selected in thePrp21p screen as multiple independent inserts, including a +1 frameshiftstarting at nucleotide 159 and containing the remainder of the ORF.Mutant prp11^(ts) cells grow at 37_(i)C when transformed with thisplasmid or with the pPL20 plasmid encoding the PRP11 wildtype gene asopposed to prp11^(ts) cells transformed with the pACTII vector (data notshown). Ylr116w was selected several times in the Mud2p screen (Table 4,FIG. 5B), including once as a −1 frameshift fusion (aa 10-362). We firstshowed that YLR116W is an essential gene: fifteen tetrads dissected froma diploid strain deleted for one YLR116W allele with a TRP1 cassetteexhibit only two viable spores each, none of them being trp⁺. Thisdiploid strain was transformed with the plasmid potentially producingthe Ylr116w frameshifted fusion protein and the resulting cells weresporulated. Ten tetrads were dissected: two and four tetrads eachexhibited 3 and 4 viable spores, respectively. The additional sporeswere trp⁺ leu⁺, demonstrating that the viability depends upon thepresence of the LEU2 plasmid carrying the frameshifted fusion gene. Inconclusion, we demonstrate that frameshift events, corresponding to a −1and a +1 reading, can occur and are indeed selected in two-hybridscreens via the production of the frameshifted fusion protein.

[0838] Discussion

[0839] Improvement and Standardization for a Rational Use of theTwo-hybrid Screen

[0840] Two-hybrid screens have been mostly used to find an appealingpartner for a given protein. Many positive clones are usually discardedbecause their sequence is not related to any known protein or becausethe identified protein has no expected link with the protein used asbait. Here we report a reproducible strategy for exhaustive two-hybridscreens. This approach is based on the multiple round screening of ayeast genomic DNA library. The methodological improvements are: i) theroutine analysis of the complete yeast genomic library for eachscreening; ii) a very strong selectivity; iii) the identification of allcandidates by sequence; iv) a classification of candidates that reflectstheir worth for a new screen without speculating on their function (newbaits chosen among A1, A2 and A3 categories); and v) the specificity ofthe identified interactions that comes out from the multiplicity of thescreens.

[0841] The strong point of this strategy is its exhaustivity. Thelibrary contains one random fusion every 4 nucleotides, allowing afairly good coverage of the genome. The library is exhaustively screenedin each mating experiment. The mating strategy is very easy, highlyreproducible, and allows controlled screening experiments to beperformed with exactly the same library. We favor the mating protocol onfilters followed by direct plating on selective medium instead ofreplica-plating diploids (Bendixen et al., 1994) or transformation sinceour protocol allows a larger number of interactions to be tested. Thiscontrasts with two-hybrid screens of yeast genomic libraries that havebeen previously reported. In most cases, a limited number ofinteractions was tested (usually between 4×104 and 4×105, Bendixen etal., 1994; He and Jacobson, 1995; Stutz et al., 1995). In theseexperiments a low coverage of the yeast genome was achieved andscreening was not exhaustive. Moreover, our results show that for preysfound as multiple independent fusions, many expected fusions were notselected, probably due to problems in expression, stability or foldingof fusion proteins. This emphasizes the importance of screening manyinteractions for recovering a set of interacting partners as complete aspossible.

[0842] The fifteeen screens presented in this study show that proteinsused as baits behave differently (FIG. 4). These data suggest that thedistribution profile in categories of preys reflects an intrinsicproperty of the bait protein. Some profiles are simpler than others; thereasons for this are so far unexplainable. We expect that increasing thenumber of screens will probably reveal common features shared byproteins exhibiting the same profile and will facilitate the choice offuture baits.

[0843] The selection of multiple independent fusions for a given preyallows the definition of an interacting domain. This is an efficient andreliable alternative strategy to the analysis of multiple deletionmutants that must otherwise be constructed (He et al., 1996; Iwabuchi etal., 1993; Rain et al., 1996). We describe also that out of framefusions are frequently selected and we show that, indeed, frameshiftedpolypeptides are produced. Both +1 and −1 frameshifts have been reportedin yeast and might occur at high levels (Stahl et al., 1995). Theproduction of out of frame fusions can be advantageous in two-hybridscreening since in frame fusions may represent dominant negative mutantsthat could be counterselected. Such an example is found with Prp11ppreys found in the Prp21p screen (Table 6, see also Fields and Jang,1996; Legrain and Chapon, 1993). Finding expressed out of frame fusiongenes emphasizes the importance of the identification of the interactingclone independently of its theoretical coding capacity, which can bestbe done in a yeast genomic library screening due to the availability ofthe complete sequence of the genome.

[0844] Multiple Rounds of Exhaustive Two-hybrid Screens EstablishPhysical Links Between Functionally Related Proteins

[0845] We are aware that the interactions defined by two-hybrid assayswould not cover all possible interactions, neither would allinteractions be directly assessed as biologically relevant. However, ourmultiple round strategy has been used successfully to identify physicallinks between several proteins implicated in nuclear pre-mRNA splicing.First, we confirm interactions between Prp9p, Prp11p and Prp21p thatwere previously demonstrated (Legrain and Chapon, 1993; Legrain et al.,1993). Secondly, new splicing factors are identified in the series ofscreens started with Cus1p, a yeast homologue of the human SAP145protein which is a component of the SF3b splicing factor (Champion andReed, 1994). We first found that Cus1p interacts with Yor319w, a yeastprotein identified as highly homologous with human SAP49, a secondcomponent of the SF3b factor shown by in vitro experiments to interactwith SAP145 (Champion and Reed, 1994). Then we identified a smallprotein in the Yor319w screen, Yjr022w, that exhibits the Sm1 and Sm2motifs found in core proteins of the snRNP particles (Hermann et al.,1995; Seraphin, 1995). A third round of screening with Yjr022w used asbait selected two additional Sm proteins, Yb1026w and Uss1p. The latterprotein has been shown to be associated with the U6 snRNP. Thus, thisseries of two-hybrid screens identifies several novel yeast splicingfactors (Yor319w, Yjr022w, Yb1026w) and suggests functional linksbetween the U2 snRNP and the U6 snRNP. The association of U2 and U6snRNPs is required in the mature spliceosome, but the molecular basis ofthis association is so far unknown. Yjr022w and Yb1026w could either becomponents of a novel complex that bridges the U2 and the U6 snRNPs orcould be U6 snRNP-associated proteins.

[0846] It has been suggested that two sets of Sm proteins are associatedwith the U1, U2, U4 and U5 snRNP, and with the U6 snRNP, respectively,each member of a set being more closely related to a given member of theother set (FIG. 6, Seraphin, 1995). Eight proteins constitute the U1-U5core particles in mammalian cells (Sm B and B′, derived from the samegene by alternative splicing, D1, D2, D3, E, F and G) whereas no humanU6-associated Sm protein has been identified so far. In yeast, at leastfive Sm proteins are found associated with the yeast U1-U5 snRNPs andare named after their strongest homology with human Sm proteins: SmD1,SmD3, SmE, SmF/SmX3 and SmG/SmX2 (Bordonn and Tarassov, 1996; Roy etal., 1995; Rymond et al., 1993; Seraphin, 1995). In addition, two otherSm proteins, Uss1p and SmX4p, are found associated with the U6 snRNP(Cooper et al., 1995; Seraphin, 1995). In order to assign Yjr022w andYb1026w to one of these two sets, we first identified all possible yeastSm proteins in the complete yeast genome database. Fifteen proteins werefound to exhibit both Sm1 and Sm2 motifs (FIG. 6). Two proteins wereidentified as the best homologues to human SmD2 (Ylr275w) and SmB(Yer029c) proteins, respectively (FIG. 6). We note that we found Yer029cin the SmD3p screen, in accord with biochemical studies that have showndirect physical interactions between the human SmB and SmD3 proteins(Raker et al., 1996). Each of the remaining yeast Sm proteins, exceptYjr022w, is more similar to one of the U1-U5 associated proteins,suggesting indeed that a set of seven proteins, including Yb1026w, mightbe associated with the U6 snRNP particle (FIG. 6). Yjr022w cannot bebest aligned with a particular member of the family, suggesting thatthis protein plays a slightly different role than other Sm proteins.

[0847] Wide Scale Two-hybrid Screens Opens the Possibility to ExploreNovel Functional Links Between Gene Products

[0848] In addition to interactions found between splicing factors, thefinding of common preys in different screens suggests additionalfunctional links. We identified interactions between Mud2p, Ylr116w,Smy2p, Lpg4p and Prp39p that might constitute a novel biochemicalcomplex. We found a novel ORF, Ynr053c, that is selected with sixproteins including Prp9p, Prp11p and Prp21p. Ynr053c is highlyhomologous (55% identity, 71% similarity) to a recently described humanprotein responsible for auto-immune responses (Racevskis et al., 1996).Many of such human proteins are implicated in RNA processing (vanVenrooij and Pruijn, 1995). We also note that well-known transcriptionalactivators (such as Swi1p) were often selected by several baits. Theselection of transcriptional activators in two-hybrid screens is acommon finding (Bartel et al., 1993) and prevents us from seriouslyconsidering these candidates as interacting proteins. However,functional links between the transcriptional machinery and theprocessing of primary transcripts certainly exist. Repeating two-hybridscreens on a wide scale will unambiguously identify the auto-activatingdomains of transcriptional activators that will be rationally discarded.Then, the characterization of true interacting domains for transcriptionfactors will become feasible.

[0849] Links are also found with proteins known to interfere with RNAmetabolism or with nucleo-cytoplasmic transport such as Los1p and Sen1por Mtr3p and Nup157p, respectively. However, the most intriguing resultmay be the selection in our two-hybrid screens of proteins that are notsupposed to be connected with pre-mRNA processing, such as thecytoplasmic translational factors Tif4631 and Tif4632 selected in thePrp11p screen. These two proteins are implicated in the binding to thecapped mRNAs and interfere with the initiation of translation. Theseinteractions are unambiguously identified in two-hybrid screens, andtheir biological relevance must now be assessed. They may representfunctional links that were unreached through classical genetics orbiochemical purification. Finally, we also identify many unknown yeastproteins as interacting partners. Specific assays can now be applied tothese new proteins in order to assess their function.

[0850] The strategy described here is fast, reliable and efficient. Itallows us to consider a wide scale analysis of yeast gene productsthrough two-hybrid screens. Without further technical improvements whichare nevertheless in progress, several tens of screens can be performedwithin a year by a single researcher. Starting with baits chosen indifferent biochemical pathways one will progressively build networks ofprotein-protein connections. This approach will constitute one of thepossible large scale analyses of the yeast genome and should ultimatelydefine an interacting matrix within the yeast proteome.

[0851] As it appears from the teachings of the specification, theinvention is not limited in scope to one or several of the abovedetailed embodiments; the present invention also embraces all thealternatives that can be performed by one skilled in the same technicalfield, without deviating from the subject or from the scope of theinstant invention. TABLE 1 Characteristics of multiple rounds oftwo-hybrid screens. Two-hybrid diploids His⁺ LacZ⁺ identified screenbiochemical entity baits^(a) (X10⁶) colonies colonies clones^(b) 1stround Sm core particle Smd1p 40 88 40 36 Smd3p 52 243 50 47 U1 snRNPSnp1p 25 700 48 46 Mud1p 35 83 10 9 Mud2p^(c) 57 17000 61 61 U2snRNPYir009w 50 1700 82 81 yeast SF3a Prp9p 40 150 66 66 Prp11p 48 5800 86 85Prp21p 58 530 66 21^(d) yeast SF3b Cus1p 60 150 15 13 2nd round prey ofSmd3p Yer029c 40 58 10 9 prey of Mud2p Ylr116w 12 800 52 50 prey ofYir009w Ylr456w 39 330 22 21 prey of Cus1p Yor319w 40 130 10 9 3rd roundprey of Yor319w Yjr022w 96 10000 74 65

[0852] TABLE 2 ORF candidates found in Gal4-Smd1p and Gal4-Smd3ptwo-hybrid screens. ORF^(a) Cat.^(b) Number^(c) LexA^(d) Gene^(e)Function^(f) Smd1p bait YBR043c A4* 2 +++ YDR088C A3* 2 + SLU7 pre-mRNAsplicing YDR389W A4 1 ++ SAC7 YGL202w A4 2 ++ YJR033c A1* 4 (2*) +++YKL188c A1 3 (2) +++ YKL205w A4 3 ++ LOS1 pre-tRNA splicing YLR440c A42 + Smd3p bait YBR114w A4 1 +++ RAD16 DNA repair YER029c A1 17 (3 & 2*)+++ YGR290w A2* 1 − YHR107c A4 1 ++ CDC12 cytokinesis YHR178w A4 1 +++YIL139c A4 2 +++ REV7 DNA polymerase subunit YKR080w A4 1 ++ MTD1NAD-dependent dehydrogenase YLL021w A1 4 (3) +++ SPA2 cell polarityYLR430w A4 1 +++ SEN1 pre-tRNA splicing YLR433c A4 1 ++ CNA1senne/threonine phosphatase 2B YMR088C A4* 1 +++

[0853] TABLE 3 ORF candidates found in Gal4-Snp1p and Gal4-Mud1ptwo-hybrid screens. ORF^(a) Cat.^(b) Number^(c) LexA^(d) Gene^(e)Function^(f) Snp1p bait YAL047c A4 1 ++ YBL063w A4 1 − KIP1 mitoticspindle YBR112c A1 3 (3) +++ SSN6 transcription YBR117c A4 1 − TKL2transketolase 2 YCR101c A4 1 ++ YDL013w A4 2 ++ HEX3 YDR110w A2/3 1 ++YER105c A4 1 ++ NUP157 nucleopore YER172c A3 1 +++ BRR2 pre-mRNAsplicing YFL016c A4 1 − MDJ1 mitochondrial heat shock protein YFR039c A41 + YGR203w A4 1 + YHR023w A4* 3 − MYO1 myosin heavy chain YHR079c A2/33 +++ IRE1 protein kinase YLR017w A4 1 ++ YLR153c A4 1 ++ ACS2acetyl-coA synthetase 2 YLR213c A2/3* 1 ++ YLR439w A4 1 ++ MRPL4mitochondrial ribosomal protein YML016c A3* 1 ++ PPZ1 serine/threoninephosphatase YMR009w A1 2 (2) ++ YMR044w A4 1 +++ YNL199c A4 1 +++ GCR2transcription YNR016c A4 1 − FAS3 acetyl-coA carboxylase YOL004w A3 4 ++SIN3 transcription YOR275c A1 2 (2) ++ YOR346w A1 2 (2) +++ REV1 YPL016wA1 2 (2) ++ SWI1 transcription YPL215w A4 1 ++ CBP3 Mud1p bait YDR110wA2 3 +++ YLR223c A4 1 +++ RRP3 pre-rRNA processing YLR449w A4* 2 ++YML016c A4 1 +++ PPZ1 serine/threonine phosphatase YNL227c A2/3 1 ++

[0854] TABLE 4 ORF candidates found in a Gal4-Mud2p two-hybrid screen.ORF^(a) Cat.^(b) Number^(c) LexA^(d) Gene^(e) Function^(f) YBR172C A2/3*3 ++ SMY2 YGL035C A2 1 ++ MIG1 transcription YLR116w A1 41 (4 & 4*) +++YLR357w A3 1 +++ YML046w A2/3 1 +++ PRP39 pre-mRNA splicing YPL016w A1 2(2) ++ SWI1 transcription YPL105c A1 9 (3 & 1*) +++ LPG4

[0855] TABLE 5 ORF candidates found in a Gal4-Yir009w (yeast U2B″)two-hybrid screen. ORF^(a) Cat.^(b) Number^(c) LexA^(d) Gene^(e)Function^(f) YAL024c A2/3 1 ++ LTE1 cell cycle YBR114w A4 1 − RAD16 DNArepair YBR136w A4 2 + MEC1 cell cycle YDL001w A4 4 ++ YDR184c A4 3 +++YHR099w A4 2 ++ YHR197w A3 1 ++ YIL143c A2 1 − SSL2 transcriptionYJL092w A3 1 +++ SRS2 DNA helicase YKL173w A4 2 ++ GIN10 YKR099w A3 1 −BAS1 transcription YLR067c A2/3 1 − PET309 mitochondrial translationYLR433c A4 1 − CNA1 serine/threonine phosphatase 2B YLR456w A1 9 (6) ++YNL036w A1 2 (2) ++ NCE3 non-classical protein export YNL091w A4 2 ++YOR011w A2 6 ++ YOR017w A4 1 − PET127 mitochondrial translation YPL016wA4 1 ++ SWI1 transcription

[0856] TABLE 6 ORF candidates found in LexA-Prp9p, Gal4-Prp11p andGal4-Prp21p two-hybrid screens. ORF^(a) Cat.^(b) Number^(c) LexA^(d)Gene^(e) Function^(f) Prp9p bait YCL060c A4* 3 nd^(g) YCL061c A4 1YDL013w A2 2 HEX3 YDR421W A3 1 YDR485c A1 5 (3) YJL203w A4 1 PRP21pre-mRNA splicing YML049c A3 2 YML104c A4 1 MDM1 intermediate filamentYMR005w A4 1 MPT1 YNR053C A2/3 1 YOR017w A2 1 PET 127 mitochondrialtranslation YOR023c A4 4 YOR191w A4 2 YPL146c A2 1 Prp11p bait YBR113wA4* 1 +++ YDR131c A4 1 ++ YDR180w A3 1 + YDR386w A1 2 (2) ++ YDR409w A41 ++ YER113c A4 4 +++ YGL049C A1 3 (2) +++ TIF4632 cytoplasmictranslation YGR162w A4 1 +++ TIF4631 cytoplasmic translation YJL146w A44 +++ IDS2 YJL203w A1 11 (4) +++ PRP21 pre-mRNA splicing YKL155c A1 5(5) +++ YLL014w A4* 1 + YMR117c A1 2 (2) +++ YMR302c A4 2 +++ RNA12pre-rRNA processing YNL023c A3 1 +++ YNR053c A2/3 3 +++ YPL215w A2 2 ++CBP3 Prp21p bait YBL067c A4 1 − UBP13 ubiquitin carboxyl-terminalhydrolase YBL101c A2* 1 ++ YCR081w A4 1 ++ SRB8 transcription YDL030w A21 +++ PRP9 pre-mRNA splicing YDL043c A1 4 (1 & 3*) +++ PRP11 pre-mRNAsplicing YDR170c A4 1 ++ SEC7 non-clathrin vesicle coat YHL007C A4 3 +++STE20 serine/threonine kinase YKR090W A4 1 +++ YLR067c A2/3 2 − PET309mitochondrial translation YMR285c A2 1 ++ YNR053c A2/3 1 ++ YOL091w A4 1+++ YOL136c A4 1 +++ PFK27 6-phosphofructose-2-kinase

[0857] TABLE 7 ORF candidates found in a Gal4-Cus1p two-hybrid screen.ORF^(a) Cat.^(b) Number^(c) LexA^(d) Gene^(e) Function^(f) YBR112c A41 + SSN6 transcription YER172c A1 4 (2) ++ BRR2 pre-mRNA splicingYGL035c A2 2 ++ MIG1 transcription YLR090w A4 2 ++ XDJ1 YOR319w A2 2 +++HSH49 YPL016w A1 2 (2) ++ SWI1 transcription

[0858] TABLE 8 ORF candidates found in second round two-hybrid screens.ORF^(a) Cat.^(b) Number^(c) LexA^(d) Gene^(e) Function^(f) Yer029c baitYDR422c A3 2 +++ SIP1 transcription YLR220w A2/3 1 ++ CCC1 YNL187W A2/3*2 +++ YNR053c A2/3 2 − Ylr456w bait YER032w A4 1 ++ YIL144w A4* 1 −YKR084c A4 1 +++ HBS1 YNR053c A2/3 3 − Ylr116w bait YBR172C A1 6 (4) +++SMY2 YDL156w A2 1 − YDL161w A4 1 ++ YER013w A3 2 ++ PRP22 pre-mRNAsplicing YIL130w A4 1 ++ YJR005w A2/3 1 ++ YAP80 clathrin-associatedcomplex YJR066w A3 1 ++ TOR1 phosphatidylinositol kinase YML046w A2* 1 −PRP39 pre-mRNA splicing YMR065w A3 1 ++ YPL016w A4 2 ++ SWI1transcription YPL105c A1 13 (3 & 5*) +++ LPG4 Yor319w bait YGR056W A4 1− YHR209W A4 3 +++ YJR022w A1 2 (2) +++ YMR240c A1 3 (2) +++ CUS1pre-mRNA splicing

[0859] TABLE 9 ORF candidates found in a third round two-hybrid screenusing a Gal4-Yjr022w bait. ORF^(a) Cat.^(b) Number^(c) LexA^(d) Gene^(e)Function^(f) YBL026w A1* 4 (1 & 2*) +++ SNP3 YBR003w A4 1 − COQ1Coenzyme Q biosynthesis YCR077c A4* 1 − YDR228c A1 2 (2) ++ YDR277c A2/31 − MTH1 transcription YEL015w A1* 5 (3*) − YER112W A2 1 +++ USS1pre-mRNA splicing YGL096w A1 2 (2) ++ YGL173c A4 1 − KEM1 RNA and DNA5′-3′ exonuclease YGR158c A2 1 +++ MTR3 mRNA transport YHR034c A4 1 +++YHR035w A4 1 ++ YIL173w A4 1 − YNL050c A4* 3 +++ YNL118c A1 10 (2 & 3*)− PSU1 YNR050c A4 1 − LYS9 lysine biosynthesis YNR053c A2/3 7 − YOR076cA3 1 ++ YOR319w A2 1 +++ HSH49

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1 29 1 25 DNA Saccharomyces cerevisiae 1 cgcgtttgga atcactacag ggatg 252 25 DNA Saccharomyces cerevisiae 2 gaaattgaga tggtgcacga tgcac 25 3 20DNA Saccharomyces cerevisiae 3 ggcttaccca tacgatgttc 20 4 16 DNASaccharomyces cerevisiae 4 atcccggacg aaggcc 16 5 13 DNA Saccharomycescerevisiae 5 ggccttcgtc cgg 13 6 79 PRT Saccharomyces cerevisiae 6 MetLeu Gln His Ile Asp Tyr Arg Met Arg Cys Ile Leu Gln Asp Gly 1 5 10 15Arg Ile Phe Ile Gly Thr Phe Lys Ala Phe Asp Lys His Met Asn Leu 20 25 30Ile Leu Cys Asp Cys Asp Glu Phe Arg Lys Ile Lys Pro Lys Asn Ser 35 40 45Lys Gln Ala Glu Arg Glu Glu Lys Arg Val Leu Gly Leu Val Leu Leu 50 55 60Arg Gly Glu Asn Leu Val Ser Met Thr Val Glu Gly Pro Pro Pro 65 70 75 779 PRT Saccharomyces cerevisiae 7 Leu Ala Asn Leu Ile Asp Tyr Lys LeuArg Val Leu Thr Gln Asp Gly 1 5 10 15 Arg Val Tyr Ile Gly Gln Leu MetAla Phe Asp Lys His Met Asn Leu 20 25 30 Val Leu Asn Glu Cys Ile Glu GluArg Val Pro Lys Thr Gln Leu Asp 35 40 45 Lys Leu Arg Pro Arg Lys Asp SerLys Asp Leu Gly Leu Thr Ile Leu 50 55 60 Arg Gly Glu Gln Ile Leu Ser ThrVal Val Glu Asp Lys Pro Leu 65 70 75 8 74 PRT Saccharomyces cerevisiae 8Ile Val Ser Ser Val Asp Arg Lys Ile Phe Val Leu Leu Arg Asp Gly 1 5 1015 Arg Met Leu Phe Gly Val Leu Arg Thr Phe Asp Gln Tyr Ala Asn Leu 20 2530 Ile Leu Gln Asp Cys Val Glu Arg Ile Tyr Phe Ser Glu Glu Asn Lys 35 4045 Tyr Ala Glu Glu Asp Arg Gly Ile Phe Met Ile Arg Gly Glu Asn Val 50 5560 Val Met Leu Gly Glu Val Asp Ile Asp Lys 65 70 9 69 PRT Saccharomycescerevisiae 9 Leu Met Lys Leu Ser His Glu Thr Val Thr Ile Glu Leu Lys AsnGly 1 5 10 15 Thr Gln Val His Gly Thr Ile Thr Gly Val Asp Val Ser MetAsn Thr 20 25 30 His Leu Lys Ala Val Lys Met Thr Leu Lys Asn Arg Glu ProVal Gln 35 40 45 Leu Glu Thr Leu Ser Ile Arg Gly Asn Asn Ile Arg Tyr PheIle Leu 50 55 60 Pro Asp Ser Leu Pro 65 10 79 PRT Saccharomycescerevisiae 10 Leu Lys Lys Leu Arg Asn Glu Gln Val Thr Ile Glu Leu LysAsn Gly 1 5 10 15 Thr Thr Val Trp Gly Thr Leu Gln Ser Val Ser Pro GlnMet Asn Ala 20 25 30 Ile Leu Thr Asp Val Lys Leu Thr Leu Pro Gln Pro ArgLeu Asn Lys 35 40 45 Leu Asn Ser Asn Gly Ile Ala Met Ala Ser Leu Gln TyrIle Asn Ile 50 55 60 Arg Gly Asn Thr Ile Arg Gln Ile Ile Leu Pro Asp SerLeu Asn 65 70 75 11 71 PRT Saccharomyces cerevisiae 11 Phe Lys Thr LeuVal Asp Gln Glu Val Val Val Glu Leu Lys Asn Asp 1 5 10 15 Ile Glu IleLys Gly Thr Leu Gln Ser Val Asp Gln Phe Leu Asn Leu 20 25 30 Lys Leu AspAsn Ile Ser Cys Thr Asp Glu Lys Lys Tyr Pro His Leu 35 40 45 Gly Ser ValArg Asn Ile Phe Ile Arg Gly Ser Thr Val Arg Tyr Val 50 55 60 Tyr Leu AsnLys Asn Met Val 65 70 12 79 PRT Saccharomyces cerevisiae 12 Gln Ser ValLys Asn Asn Thr Gln Val Leu Ile Asn Cys Arg Asn Asn 1 5 10 15 Lys LysLeu Leu Gly Arg Val Lys Ala Phe Asp Arg His Cys Asn Met 20 25 30 Val LeuGlu Asn Val Lys Glu Met Trp Thr Glu Val Pro Lys Ser Gly 35 40 45 Lys GlyLys Lys Lys Ser Lys Pro Val Asn Ile Ser Lys Met Phe Leu 50 55 60 Arg GlyAsp Ser Val Ile Val Val Leu Arg Asn Pro Leu Ile Ala 65 70 75 13 75 PRTSaccharomyces cerevisiae 13 Asp Ala Met Val Thr Arg Thr Pro Val Ile IleSer Leu Arg Asn Asn 1 5 10 15 His Lys Ile Ile Ala Arg Val Lys Ala PheAsp Arg His Cys Asn Met 20 25 30 Val Leu Glu Asn Val Lys Glu Leu Trp ThrGlu Lys Lys Gly Lys Asn 35 40 45 Val Ile Asn Arg Glu Arg Phe Ile Ser LysLeu Phe Leu Arg Gly Asp 50 55 60 Ser Val Ile Val Val Leu Lys Thr Pro ValGlu 65 70 75 14 76 PRT Saccharomyces cerevisiae 14 Leu Lys Leu Asn LeuAsp Glu Arg Val Tyr Ile Lys Leu Arg Gly Ala 1 5 10 15 Arg Thr Leu ValGly Thr Leu Gln Ala Phe Asp Ser His Cys Asn Ile 20 25 30 Val Leu Ser AspAla Val Glu Thr Ile Tyr Gln Leu Asn Asn Glu Glu 35 40 45 Leu Ser Glu SerGlu Arg Arg Cys Glu Met Val Phe Ile Arg Gly Asp 50 55 60 Thr Val Thr LeuIle Ser Thr Pro Ser Glu Asp Asp 65 70 75 15 69 PRT Saccharomycescerevisiae 15 Leu His Glu Ala Glu Gly His Ile Val Thr Cys Glu Thr AsnThr Gly 1 5 10 15 Glu Val Tyr Arg Gly Lys Leu Thr Glu Ala Glu Asp AsnMet Asn Cys 20 25 30 Gln Met Ser Asn Ile Thr Val Thr Tyr Arg Asp Gly ArgVal Ala Gln 35 40 45 Leu Glu Gln Val Tyr Ile Arg Gly Ser Lys Ile Arg PheLeu Ile Leu 50 55 60 Pro Asp Met Leu Lys 65 16 69 PRT Saccharomycescerevisiae 16 Leu Asn Glu Ala Gln Gly His Ile Val Ser Leu Glu Leu ThrThr Gly 1 5 10 15 Ala Thr Tyr Arg Gly Lys Leu Val Glu Ser Glu Asp SerMet Asn Val 20 25 30 Gln Leu Arg Asp Val Ile Ala Thr Glu Pro Gln Gly AlaVal Thr His 35 40 45 Met Asp Gln Ile Phe Val Arg Gly Ser Gln Ile Lys PheIle Val Val 50 55 60 Pro Asp Leu Leu Lys 65 17 80 PRT Saccharomycescerevisiae 17 Leu Thr Asn Ala Lys Gly Gln Gln Met Gln Ile Glu Leu LysAsn Gly 1 5 10 15 Glu Ile Ile Gln Gly Ile Leu Thr Asn Val Asp Asn TrpMet Asn Leu 20 25 30 Thr Leu Ser Asn Val Thr Glu Tyr Ser Glu Glu Ser AlaIle Asn Ser 35 40 45 Glu Asp Asn Ala Glu Ser Ser Lys Ala Val Lys Leu AsnGlu Ile Tyr 50 55 60 Ile Arg Gly Thr Phe Ile Lys Phe Ile Lys Leu Gln AspAsn Ile Ile 65 70 75 80 18 68 PRT Saccharomyces cerevisiae 18 Leu GlnAsn Arg Ser Arg Ile Gln Val Trp Leu Tyr Glu Gln Val Asn 1 5 10 15 MetArg Ile Glu Gly Cys Ile Ile Gly Phe Asp Glu Tyr Met Asn Leu 20 25 30 ValLeu Asp Asp Ala Glu Glu Ile His Ser Lys Thr Lys Ser Arg Lys 35 40 45 GlnLeu Gly Arg Ile Met Leu Lys Gly Asp Asn Ile Thr Leu Leu Gln 50 55 60 SerVal Ser Asn 65 19 74 PRT Saccharomyces cerevisiae 19 Leu Gln Gln Gln ThrPro Val Thr Ile Trp Leu Phe Glu Gln Ile Gly 1 5 10 15 Ile Arg Ile LysGly Lys Ile Val Gly Phe Asp Glu Phe Met Asn Val 20 25 30 Val Ile Asp GluAla Val Glu Ile Pro Val Asn Ser Ala Asp Gly Lys 35 40 45 Glu Asp Val GluLys Gly Thr Pro Leu Gly Lys Ile Leu Leu Lys Gly 50 55 60 Asp Asn Ile ThrLeu Ile Thr Ser Ala Asp 65 70 20 77 PRT Saccharomyces cerevisiae 20 IleAsp Lys Thr Ile Asn Gln Lys Val Leu Ile Val Leu Gln Ser Asn 1 5 10 15Arg Glu Phe Glu Gly Thr Leu Val Gly Phe Asp Asp Phe Val Asn Val 20 25 30Ile Leu Glu Asp Ala Val Glu Trp Leu Ile Asp Pro Glu Asp Glu Ser 35 40 45Arg Asn Glu Lys Val Met Gln His His Gly Arg Met Leu Leu Ser Gly 50 55 60Asn Asn Ile Ala Ile Leu Val Pro Gly Gly Lys Lys Thr 65 70 75 21 70 PRTSaccharomyces cerevisiae 21 Leu Asn Gly Leu Thr Gly Lys Pro Val Met ValLys Leu Lys Trp Gly 1 5 10 15 Met Glu Tyr Lys Gly Tyr Leu Val Ser ValAsp Gly Tyr Met Asn Met 20 25 30 Gln Leu Ala Asn Thr Glu Glu Tyr Ile AspGly Ala Leu Ser Gly His 35 40 45 Leu Gly Glu Val Leu Ile Arg Cys Asn AsnVal Leu Tyr Ile Arg Gly 50 55 60 Val Glu Glu Glu Glu Glu 65 70 22 68 PRTSaccharomyces cerevisiae 22 Leu Lys Gly Leu Val Asn His Arg Val Gly ValLys Leu Lys Phe Asn 1 5 10 15 Ser Thr Glu Tyr Arg Gly Thr Leu Val SerThr Asp Asn Tyr Phe Asn 20 25 30 Leu Gln Leu Asn Glu Ala Glu Glu Phe ValAla Gly Val Ser His Gly 35 40 45 Thr Leu Gly Glu Ile Phe Ile Arg Cys AsnAsn Val Leu Tyr Ile Arg 50 55 60 Glu Leu Pro Asn 65 23 71 PRTSaccharomyces cerevisiae 23 Leu Ser Asp Ile Ile Gly Lys Thr Val Asn ValLys Leu Ala Ser Gly 1 5 10 15 Leu Leu Tyr Ser Gly Arg Leu Glu Ser IleAsp Gly Phe Met Asn Val 20 25 30 Ala Leu Ser Ser Ala Thr Glu His Tyr GluSer Asn Asn Asn Lys Leu 35 40 45 Leu Asn Lys Phe Asn Ser Asp Val Phe LeuArg Gly Thr Gln Val Met 50 55 60 Tyr Ile Ser Glu Gln Lys Ile 65 70 24 68PRT Saccharomyces cerevisiae 24 Leu Lys Lys Phe Met Asp Lys Lys Leu SerLeu Lys Leu Asn Gly Gly 1 5 10 15 Arg His Val Gln Gly Ile Leu Arg GlyPhe Asp Pro Phe Met Asn Leu 20 25 30 Val Ile Asp Glu Cys Val Glu Met AlaThr Ser Gly Gln Gln Asn Asn 35 40 45 Ile Gly Met Val Val Ile Arg Gly AsnSer Ile Ile Met Leu Glu Ala 50 55 60 Leu Glu Arg Val 65 25 71 PRTSaccharomyces cerevisiae 25 Leu Lys Lys Tyr Met Asp Lys Lys Ile Leu LeuAsn Ile Asn Gly Ser 1 5 10 15 Arg Lys Val Ala Gly Ile Leu Arg Gly TyrAsp Ile Phe Leu Asn Val 20 25 30 Val Leu Asp Asp Ala Met Glu Ile Asn GlyGlu Asp Pro Ala Asn Asn 35 40 45 His Gln Leu Gly Leu Gln Thr Val Ile ArgGly Asn Ser Ile Ile Ser 50 55 60 Leu Glu Ala Leu Asp Ala Ile 65 70 26 81PRT Saccharomyces cerevisiae 26 Leu Ala Lys Tyr Lys Asp Ser Lys Ile ArgVal Lys Leu Met Gly Gly 1 5 10 15 Lys Leu Val Ile Gly Val Leu Lys GlyTyr Asp Gln Leu Met Asn Leu 20 25 30 Val Leu Asp Asp Thr Val Glu Tyr MetSer Asn Pro Asp Asp Glu Asn 35 40 45 Asn Thr Glu Leu Ile Ser Lys Asn AlaArg Lys Leu Gly Leu Thr Val 50 55 60 Ile Arg Gly Thr Ile Leu Val Ser LeuSer Ser Ala Glu Gly Ser Asp 65 70 75 80 Val 27 67 PRT Saccharomycescerevisiae 27 Leu Lys Asp Tyr Leu Asn Lys Arg Val Val Ile Ile Lys ValAsp Gly 1 5 10 15 Glu Cys Leu Ile Ala Ser Leu Asn Gly Phe Asp Lys AsnThr Asn Leu 20 25 30 Phe Ile Thr Asn Val Phe Asn Arg Ile Ser Lys Glu PheIle Cys Lys 35 40 45 Ala Gln Leu Leu Arg Gly Ser Glu Ile Ala Leu Val GlyLeu Ile Asp 50 55 60 Ala Glu Asn 65 28 114 DNA Saccharomyces cerevisiae28 gagagtagta acaaaggtca aagacagttg actgtatcgc cggaatttat ggccatggag 60gccccgggga tccgtcgacc tgcagccaag ctaattccgg gcgaatttct tatg 114 29 288DNA Saccharomyces cerevisiae 29 cgcgtttgga atcactacag ggatgtttaataccactaca atggatgatg tatataacta 60 tctattcgat gatgaagata ccccaccaaacccaaaaaaa gagatctgta tggcttaccc 120 atacgatgtt ccagattacg ctagcttgggtggtcatatg gccatggagg ccccggggat 180 ccgaattcga gctcgactag ctagctgactcgagagatct atgaatcgta gatactgaaa 240 aaccccgcaa gttcacttca actgtgcatcgtgcaccatc tcaatttc 288

What is claimed is:
 1. A method for selecting a polynucleotide encodinga prey polypeptide, said prey polypeptide being able to interact with abait polypeptide, comprising the steps of: a) subjecting a baitpolynucleotide encoding the bait polypeptide, to a two-hybrid screeningmethod, wherein said two-hybrid screening method comprises a step ofmating at least one first recombinant yeast cell containing the preypolynucleotide to be assayed with a second haploid recombinant yeastcell containing the bait polynucleotide, provided that one haploid yeastcell among the first recombinant yeast cell or the second recombinantyeast cell also contains at least one detectable gene that is activatedby a polypeptide including a transcriptional activation domain; b)selecting the recombinant diploid yeast cell obtained at step a) forwhich the detectable gene has been expressed to a degree greater thanexpression in the absence of interaction between the bait polypeptideand the prey polypeptide; and c) optionally characterizing the preypolynucleotide contained in each diploid yeast cell selected at step b).2. The method according to claim 1, further comprising repeating atleast once steps a) to c) with the previously characterized preypolynucleotide used as the bait polynucleotide.
 3. The method accordingto claim 2, wherein the number of repeats of steps a) to c) with thepreviously characterized prey polynucleotide used as the baitpolynucleotide is in the range from 1 to 10, preferably from 1 to 5 andmost preferably
 3. 4. The method according to claim 1, wherein the baitpolynucleotide used that corresponds to a selected prey belongs to thefollowing group of polynucleotide consisting in: a) a polynucleotidethat is identical to said selected prey polynucleotide; b) apolynucleotide containing the complete ORF including said selected preypolynucleotide; c) a polynucleotide which is any polynucleotide fragmentcomprised in the complete ORF including said selected preypolynucleotide.
 5. The method according to claim 1, wherein the matingstep is performed on a filter.
 6. The method according to claim 5,wherein the mating step is performed during a maximum of 5 hours.
 7. Themethod according to claim 5, wherein the efficiency value of the matingstep is up to 50%.
 8. The method according to claim 1, wherein thedetectable gene is LacZ, HIS3 or both LacZ and HIS3.
 9. The methodaccording to claim 1, wherein the prey polynucleotide is containedwithin the plasmid pACTIIst.
 10. The method according to claim 1,wherein the bait polynucleotide is contained within the plasmid pAS2ΔΔ.11. The method according to claim 1, wherein the prey polynucleotide isprovided by a DNA library.
 12. The method according to claim 11, whereinthe DNA library has been prepared from the genome or from the mRNA of aprokaryotic host organism.
 13. The method according to claim 11, whereinthe DNA library has been prepared from the genome or from the mRNA of aeukaryotic host organism.
 14. The method according to claim 13, whereinthe DNA library has been prepared from the genomic DNA of Saccharomycescerevisiae.
 15. The method according to claim 14 wherein the DNA libraryis the FRYL library contained in the collection of recombinant E. colistrain MR32 cells that have been deposited at the Collection Nationalede Culture de Microorganismes (C.N.C.M.) on Dec. 21, 1995 under theaccess number I-1651.
 16. The method according to claim 1, wherein theprey polynucleotide selected at step b) is classified within one of thefollowing prey polynucleotide classes: a) a polynucleotide contained inan intergenic region or on the reverse orientation of an ORF containedin the genome of the organism from which the initial DNA library hasbeen prepared (B). b) a polynucleotide selected several times andcontained in different clones of the initial DNA library (A1); c) apolynucleotide selected only once and having a 5′ end of the codingstrand starting close to an initiation codon of an ORF contained in thegenome of the organism from which the initial DNA library has beenprepared (A2); d) a polynucleotide selected only once and having anucleotide length of at least 1000 bases (A3); e) a polynucleotideselected only once and having characteristics different from thepolynucleotides of the a), b), c) and d) classes (A4).
 17. Therecombinant plasmid pACTIIst.
 18. The recombinant plasmid pAS2ΔΔ.
 19. Arecombinant host organism containing the recombinant plasmid pACTIIst.20. A recombinant host organism containing the recombinant plasmidpAS2ΔΔ.
 21. The recombinant host organism according to claim 19 or 20,which is chosen among the group consisting in a E. coli and a yeastcell.
 22. A collection of recombinant cell clones from a host organismaccording to claim 19, each different cell clone containing an insertedpolynucleotide from a DNA library.
 23. The collection of recombinantcell clones according to claim 22, wherein the DNA library is preparedfrom the genomic DNA of Saccharomyces cerevisiae.
 24. The collection ofrecombinant cell clones according to claim 23, wherein the number ofdifferent cell clones is about 15×10⁶.
 25. The collection of recombinantcell clones according to claim 24, which is the FRYL library containedin the collection of recombinant E. coli strain MR32 cells that havebeen deposited at the Collection Nationale de Culture de Microorganismes(C.N.C.M.) on Dec. 21, 1995 under the access number I-1651.
 26. Arecombinant diploid yeast cell selected by the method according toclaim
 1. 27. A polynucleotide that has been selected with the methodaccording claim
 1. 28. A polypeptide that is encoded by a polynucleotideaccording to claim
 27. 29. A kit for selecting at least one polypeptideof interest belonging to Saccharomyces cerevisiae, wherein said kitcomprises: a) at least one complete collection of yeast cell clonescontaining the whole polynucleotide inserts representative of the genomefrom a prokaryotic or eukaryotic organism having a compact genome; b)optionally the plasmid pAS2ΔΔ; c) optionally the plasmid pACTIIst; d)optionally, a haploid yeast cell to be transformed with a plasmidcontaining a bait polynucleotide of interest; and e) optionally, thereagents necessary to visualize the expression of at least onedetectable gene, such as X-Gal.
 30. A kit according to claim 29, whereinthe genomic DNA library is the FRYL library.
 31. A computer useablemedium containing computer readable data related to the interactionsbetween at least one bait polypeptide and at least one prey polypeptideencoded at least in part by a prey polynucleotide that has been selectedwith the method according to claim 1.